Dendrimer like amino amides possessing sodium channel blocker activity for the treatment of dry eye and other mucosal diseases

ABSTRACT

Sodium channel blockers represented by the formula: 
     
       
         
         
             
             
         
       
     
     are provided where the structural variables are defined herein. The invention also includes a variety of compositions, combinations and methods of treatment using these inventive sodium channel blockers.

CONTINUING APPLICATION INFORMATION

This application is a Continuation of U.S. application Ser. No.14/564,299, filed Dec. 9, 2014, which is a Continuation of U.S.application Ser. No. 13/904,634, filed on May 29, 2013, which claimsbenefit of the filing date of Provisional Application Ser. No.61/652,481, filed on May 29, 2012, and incorporated by reference in itsentirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to sodium channel blockers. The presentinvention also includes a variety of methods of treatment using theseinventive sodium channel blockers, their pharmaceutically acceptablesalt forms, which are useful as sodium channel blockers, compositionscontaining the same, therapeutic methods including but not limited totreating dry eye, treating Sjogren's disease-associated dry eye,promoting ocular hydration, promoting corneal hydration and thetreatment of other mucousal diseases and uses for the same and processesfor preparing the same. The present invention also relates to novelcompounds for the treatment of dry eye, particularly including(2R,2′R)—N,N′-(3,3′-(2-(4-(4-(3-(3,5-diamino-6-chloropyrazine-2-carbonyl)guanidino)butyl)phenoxy)ethylazanediyl)bis(propane-3,1-diyl))bis(2-amino-6-guanidinohexanamide)and its pharmaceutically acceptable salt forms, which are useful assodium channel blockers, compositions containing the same, therapeuticmethods including but not limited to treating dry eye, treatingSjogren's disease-associated dry eye, promoting ocular hydration,promoting corneal hydration and the treatment of other mucousal diseasesand uses for the same and processes for preparing the same.

2. Description of the Background

The mucosal epithelial cells at the interface between the environmentand the body have evolved a number of “innate defenses”, i.e.,protective mechanisms. A principal function of such innate defense is tocleanse these surfaces from microorganisms, particles and other foreignmaterial. This process requires the presence of a layer of liquid topropel these microorganisms, particles and other foreign material awayfrom the body to avoid colonization of microorganisms and/or tissuedamage. Typically, the quantity of the liquid layer on a mucosal surfacereflects the balance between epithelial liquid secretion, oftenreflecting anion (Cl⁻ and/or HCO₃ ⁻) secretion coupled with water (and acation counter-ion), and epithelial liquid absorption, often reflectingNa⁺ absorption, coupled with water and counter anion (Cl⁻ and/or HCO₃⁻). Many diseases of mucosal surfaces are caused by too littleprotective liquid on those mucosal surfaces created by an imbalancebetween secretion (too little) and absorption (relatively too much). Thecritical salt transport processes that characterize a number of mucosaldysfunctions resides in the epithelial layer of the mucosal surface.

Chronic dry eye disease, also known as keratoconjunctivitis sicca, isone of the most frequently diagnosed ocular diseases, affecting morethan 5 million people in the United States alone. Dry eye ischaracterized by inadequate aqueous tear fluid on the eyes, resulting inpainful irritation, inflammation on the ocular surface, and impairedvision, and is caused by failure of lacrimal glands to secrete liquid inthe face of continued Na⁺ dependent liquid absorption on conjunctivalsurfaces. Dry eye is a multi-factorial disease, resulting from a commonetiology of insufficient tear film, causing ocular surface damage andsymptoms of ocular discomfort.

The few current therapies available, which include bothimmunosuppressive agents and over-the-counter tear replacements, are notsufficiently efficacious for many users or only provide transient relieffrom dry eye symptoms. The dry eye market is dominated byover-the-counter (OTC) tear replacements or artificial tears, estimatedto be used by ˜80% of dry eye patients. Artificial tears provideimmediate symptomatic relief from the sensation of ocular burning andirritation by adding liquid to the ocular surface. Yet, the benefitsfrom artificial tears are short-lived as the fluid drops are rapidlycleared from the ocular surface, providing, at most, palliative reliefand requiring frequent application throughout the day.

While individuals with dry eye may not exhibit overt ocular inflammationsuch as red, inflamed eyes, chronic ocular inflammation is now wellrecognized as a significant factor perpetuating the chronic cycle of dryeye. The one approved prescription drug for the treatment of chronic dryeye is Restasis® (0.05% Cyclosporine A emulsion, Allergan), which ismarketed to increase tear output “in patients whose tear production ispresumed to be suppressed due to ocular inflammation associated withkeratoconjunctivitis sicca.” In a six month, Phase 3 pivotal trial insubjects with dry eye, Restasis statistically increased tear volume(assessed by Schirmer wetting) in 15% of the treated individuals,compared to 5% on vehicle. While the mechanism of Restasis is not fullyunderstood, it is speculated that the inhibition of chronic ocularinflammation may, over time, restore corneal sensitivity and improvereflex tearing. However, Restasis has a low responder rate, a 3 monthdelay for full therapeutic benefit, and side effects, such as burning onapplication.

Therefore, the development of novel hydrating agents to treat dry eyewould be of tremendous benefit to the therapeutic milieu. The volume oftear film on the ocular surface represents a balance between tear fluidoutput versus fluid loss via drainage, evaporation, or epithelialabsorption. Similar to other epithelial tissues, the epithelium of theconjunctiva and cornea are capable of regulating the hydration status ofthe mucosal surface through active salt and water transport.

One approach to replenish the protective liquid layer on mucosalsurfaces is to “re-balance” the system by blocking Na⁺ channel andliquid absorption. The epithelial protein that mediates therate-limiting step of Na⁺ and liquid absorption is the epithelial Na⁺channel (ENaC) and is a key regulator of sodium (and water) absorptionin numerous tissues, including the eye. ENaC is expressed on the apicalsurface of the corneal and conjunctival epithelia in rodents, largermammals, and man where it functions as a critical pathway for sodium(and water) absorption (Krueger B, Schlötzer-Schrehardt U, Haerteis S,Zenkel M, Chankiewitz V E, Amann K U, Kruse F E, Korbmacher C. Foursubunits (αβγδ) of the epithelial sodium channel (ENaC) are expressed inthe human eye in various locations. Invest Ophthalmol Vis Sci. 2012;53(2):596-604).

In a series of in vivo bioelectric studies, Levin et al. (Levin M H, KimJ K, Hu J, Verkman A S. Potential difference measurements of ocularsurface Na+ absorption analyzed using an electrokinetic model. InvestOpthalmol Vis Sci. 2006; 47(1):306-16) confirmed ENaC-mediated sodiumtransport is a substantial contributor to the ocular surface electricalpotential difference Furthermore, the topical addition of the ENaCblocker amiloride produced an approximate doubling of tear volume thatremained elevated for >60 minutes post-administration in rats (Yu D,Thelin W R, Rogers T D, Stutts M J, Randell S H, Grubb B R, Boucher R C.Regional differences in rat conjunctival ion transport activities. Am JPhysiol Cell Physiol. 2012; 303(7):C767-80.) and rabbits (Hara S, HazamaA, Miyake M, Kojima T, Sasaki Y, Shimazaki J, Dogru M and Tsubota K. TheEffect of Topical Amiloride Eye Drops on Tear Quantity in Rabbits.Molecular Vision 2010; 16:2279-2285).

Taken together, these data provide an important proof-of-concept thatthe inhibition of ENaC will increase tear volume. The inhibition of ENaCin the eye is predicted to preserve lacrimal secretions and maintainhydration on the ocular surface. Because ENaC is positioned on theapical surface of the epithelium, i.e. the mucosal surface-environmentalinterface, to inhibit ENaC mediated Na⁺ and liquid absorption, an ENaCblocker of the amiloride class (which blocks from the extracellulardomain of ENaC) must be delivered to the mucosal surface and,importantly, be maintained at this site, to achieve therapeutic utility.The present invention describes diseases characterized by too littleliquid on mucosal surfaces and “topical” sodium channel blockersdesigned to exhibit the increased potency, reduced mucosal absorption,and slow dissociation (“unbinding” or detachment) from ENaC required fortherapy of these diseases.

The use of ENaC blockers has been reported for a variety of diseaseswhich are ameliorated by increased mucosal hydration. In particular, theuse of ENaC blockers in the treatment of respiratory diseases such aschronic bronchitis (CB), cystic fibrosis (CF), and COPD, which reflectthe body's failure to clear mucus normally from the lungs and ultimatelyresult in chronic airway infection, has been reported. See, Evidence forairway surface dehydration as the initiating event in CF airway disease,R. C. Boucher, Journal of Internal Medicine, Vol. 261, Issue 1, January2007, pages 5-16; and Cystic fibrosis: a disease of vulnerability toairway surface dehydration, R. C. Boucher, Trends in Molecular Medicine,Vol. 13, Issue 6, June 2007, pages 231-240.

Data indicate that the initiating problem in both chronic bronchitis andcystic fibrosis is the failure to clear mucus from airway surfaces. Thefailure to clear mucus reflects an imbalance in the quantities of mucusas airway surface liquid (ASL) on airway surfaces. This imbalanceresults in a relative reduction in ASL which leads to mucusconcentration, reduction in the lubricant activity of the periciliaryliquid (PCL), mucus adherence to the airway surface, and failure toclear mucus via ciliary activity to the mouth. The reduction in mucusclearance leads to chronic bacterial colonization of mucus adherent toairway surfaces. The chronic retention of bacteria, inability of localantimicrobial substances to kill mucus-entrapped bacteria on a chronicbasis, and the consequent chronic inflammatory response to this type ofsurface infection, are manifest in chronic bronchitis and cysticfibrosis.

Chronic obstructive pulmonary diseases are characterized by dehydrationof airway surfaces and the retention of mucous secretions in the lungs.Examples of such diseases include cystic fibrosis, chronic bronchitis,and primary or secondary ciliary dyskinesia. Such diseases affectapproximately 15 million patients in the United States, and are thesixth leading cause of death. Other airway or pulmonary diseasescharacterized by the accumulation of retained mucous secretions includesinusitis (an inflammation of the paranasal sinuses associated withupper respiratory infection) and pneumonia.

Chronic bronchitis (CB), including the most common lethal genetic formof chronic bronchitis, cystic fibrosis (CF), are diseases that reflectthe body's failure to clear mucus normally from the lungs, whichultimately produces chronic airways infection. In the normal lung, theprimary defense against chronic intrapulmonary airways infection(chronic bronchitis) is mediated by the continuous clearance of mucusfrom bronchial airway surfaces. This function in health effectivelyremoves from the lung potentially noxious toxins and pathogens. Recentdata indicate that the initiating problem, i.e., the “basic defect,” inboth CB and CF is the failure to clear mucus from airway surfaces. Thefailure to clear mucus reflects an imbalance between the amount ofliquid and mucin on airway surfaces. This “airway surface liquid” (ASL)is primarily composed of salt and water in proportions similar to plasma(i.e., isotonic). Mucin macromolecules organize into a well-defined“mucus layer” which normally traps inhaled bacteria and is transportedout of the lung via the actions of cilia which beat in a watery, lowviscosity solution termed the “periciliary liquid” (PCL). In the diseasestate, there is an imbalance in the quantities of mucus as ASL on airwaysurfaces. This results in a relative reduction in ASL which leads tomucus concentration, reduction in the lubricant activity of the PCL, anda failure to clear mucus via ciliary activity to the mouth. Thereduction in mechanical clearance of mucus from the lung leads tochronic bacterial colonization of mucus adherent to airway surfaces. Itis the chronic retention of bacteria, the failure of local antimicrobialsubstances to kill mucus-entrapped bacteria on a chronic basis, and theconsequent chronic inflammatory responses of the body to this type ofsurface infection, that lead to the syndromes of CB and CF.

The current afflicted population in the U.S. is 12,000,000 patients withthe acquired (primarily from cigarette smoke exposure) form of chronicbronchitis and approximately 30,000 patients with the genetic form,cystic fibrosis. Approximately equal numbers of both populations arepresent in Europe. In Asia, there is little CF but the incidence of CBis high and, like the rest of the world, is increasing.

There is currently a large, unmet medical need for products thatspecifically treat CB and CF at the level of the basic defect that causethese diseases. The current therapies for chronic bronchitis and cysticfibrosis focus on treating the symptoms and/or the late effects of thesediseases. Thus, for chronic bronchitis, β-agonists, inhaled steroids,anti-cholinergic agents, and oral theophyllines and phosphodiesteraseinhibitors are all in development. However, none of these drugs treateffectively the fundamental problem of the failure to clear mucus fromthe lung. Similarly, in cystic fibrosis, the same spectrum ofpharmacologic agents is used. These strategies have been complemented bymore recent strategies designed to clear the CF lung of the DNA(“Pulmozyme”; Genentech) that has been deposited in the lung byneutrophils that have futilely attempted to kill the bacteria that growin adherent mucus masses and through the use of inhaled antibiotics(“TOBI”) designed to augment the lungs' own killing mechanisms to ridthe adherent mucus plaques of bacteria. A general principle of the bodyis that if the initiating lesion is not treated, in this case mucusretention/obstruction, bacterial infections became chronic andincreasingly refractory to antimicrobial therapy. Thus, a major unmettherapeutic need for both CB and CF lung diseases is an effective meansof re-hydrating airway mucus (i.e., restoring/expanding the volume ofthe ASL) and promoting its clearance, with bacteria, from the lung.

R. C. Boucher, in U.S. Pat. No. 6,264,975, describes the use ofpyrazinoylguanidine sodium channel blockers for hydrating mucosalsurfaces. These compounds, typified by the well-known diureticsamiloride, benzamil, and phenamil, are effective. However, thesecompounds suffer from the significant disadvantage that they are (1)relatively impotent, which is important because the mass of drug thatcan be inhaled by the lung is limited, (2) rapidly absorbed, whichlimits the half-life of the drug on the mucosal surface; and (3) arefreely dissociable from ENaC. The sum of these disadvantages embodied inthese well-known diurectics produces compounds with insufficient potencyand/or effective half-life on mucosal surfaces to have therapeuticbenefit for hydrating mucosal surfaces.

R. C. Boucher, in U.S. Pat. No. 6,926,911, suggests the use of therelatively impotent sodium channel blockers such as amiloride, withosmolytes for the treatment of airway diseases. This combination givesno practical advantage over either treatment alone and is clinically notuseful (see Donaldson et al, N Eng J Med 2006; 353:241-250). Amiloridewas found to block the water permeability of airways and negate thepotential benefit of concurrent use of hypertonic saline and amiloride.

U.S. Pat. No. 5,817,028 to Anderson describes a method for theprovocation of air passage narrowing (for evaluating susceptibility toasthma) and/or the induction of sputum in subjects via the inhalation ofmannitol. It is suggested that the same technique can be used to inducesputum and promote mucociliary clearance. Substances suggested includesodium chloride, potassium chloride, mannitol and dextrose.

Clearly, what is needed are drugs that are more effective at restoringthe clearance of mucus from the lungs of patients with CB/CF. The valueof these new therapies will be reflected in improvements in the qualityand duration of life for both the CF and the CB populations.

Other mucosal surfaces in and on the body exhibit subtle differences inthe normal physiology of the protective surface liquids on theirsurfaces but the pathophysiology of disease reflects a common theme,i.e., too little protective surface liquid. For example, in xerostomia(dry mouth) the oral cavity is depleted of liquid due to a failure ofthe parotid sublingual and submandibular glands to secrete liquiddespite continued Na+(ENaC) transport mediated liquid absorption fromthe oral cavity.

In rhinosinusitis, there is an imbalance, as in CB, between mucinsecretion and relative ASL depletion. Finally, in the gastrointestinaltract, failure to secrete Cl— (and liquid) in the proximal smallintestine, combined with increased Na⁺ (and liquid) absorption in theterminal ileum leads to the distal intestinal obstruction syndrome(DIOS). In older patients excessive Na⁺ (and volume) absorption in thedescending colon produces constipation and diverticulitis.

The published literature includes a number of patent applications andgranted patents to Parion Sciences Inc., directed towardpyrazinoylguanidine analogs as sodium channel blockers. Examples of suchpublications include PCT Publication Nos. WO2007146867, WO2003/070182,WO2003/070184, WO2004/073629, WO2005/025496, WO2005/016879,WO2005/018644, WO2006/022935, WO2006/023573, WO2006/023617,WO2007/018640, WO2007146870, WO2007/146869, WO2008030217, WO2008/031028,WO2008/031048, WO2013/003386, WO2013/003444, and U.S. Pat. Nos.6,858,614, 6,858,615, 6,903,105, 6,995,160, 7,026,325, 7,030,117,7,064,129, 7,186,833, 7,189,719, 7,192,958, 7,192,959, 7,192,960,7,241,766, 7,247,636, 7,247,637, 7,317,013, 7,332,496, 7,368,447,7,368,450, 7,368,451, 7,375,102, 7,375,107, 7,388,013, 7,399,766,7,410,968, 7,807,834, 7,820,678, 7,842,697, 7,868,010, 7,956,059,7,981,898, 8,008,494, 8,022,210, 8,058,278, 8,124,607, 8,143,256,8,163,758, 8,198,286, 8,211,895, 8,198,286, 8,227,474, and 8,324,218.

There remains a need for novel sodium channel blocking compounds withenhanced potency and effectiveness on mucosal tissues, especially oculartissues. There also remains the need for novel sodium channel blockingcompounds that provide therapeutic effect, but minimize or eliminate theonset or progression of hyperkalemia in recipients.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide compounds that aremore potent and/or absorbed less rapidly from mucosal surfaces, such asocular surfaces, and/or are less reversible as compared to knowncompounds.

It is another aspect of the present invention to provide compounds thatare more potent and/or absorbed less rapidly and/or exhibit lessreversibility, as compared to compounds such as amilorde, benzamil, andphenamil. Therefore, the compounds will give a prolonged pharmacodynamichalf-life on mucosal surfaces as compared to known compounds.

It is another object of the present invention to provide compounds which(1) are absorbed less rapidly from mucosal surfaces, especially ocularsurfaces, as compared to known compounds and (2) when absorbed frommucosal surfaces after administration to the these surfaces, areexcreted mainly non-renally in order to minimize the chances ofhyperkalemia.

It is another object of the present invention to provide compounds whichare (1) absorbed less rapidly from mucosal surfaces, especially ocularsurfaces, as compared to known compounds and (2) are converted in vivointo metabolic derivatives thereof which have reduced efficacy inblocking sodium channels as compared to the administered parent compoundin order to minimize the chances of hyperkalemia.

It is another object of the present invention to provide compounds thatare more potent and/or absorbed less rapidly and/or exhibit lessreversibility, as compared to compounds such as amiloride, benzamil, andphenamil. Therefore, such compounds will give a prolongedpharmacodynamic half-life on mucosal surfaces as compared to previouscompounds.

It is another object of the present invention to provide compounds thatare metabolically stable. Therefore, such compounds will give aprolonged pharmacodynamic half-life on mucosal surfaces as compared toprevious compounds.

It is another object of the present invention to provide methods oftreatment that take advantage of the pharmacological properties of thecompounds described above.

In particular, it is an object of the present invention to providemethods of treatment which rely on rehydration of mucosal surfaces.

In particular, it is an object of the present invention to providemethods of treating dry eye and related ocular diseases.

The objects of the present invention may be accomplished with a class ofpyrazinoylguanidine represented by a compound of formula (I):

and includes racemates, enantiomers, diastereomers, tautomers,polymorphs, pseudopolymorphs and pharmaceutically acceptable salts,thereof, wherein:

X is hydrogen, halogen, trifluoromethyl, lower alkyl, unsubstituted orsubstituted phenyl, lower alkyl-thio, phenyl-lower alkyl-thio, loweralkyl-sulfonyl, or phenyl-lower alkyl-sulfonyl;

Y is hydrogen, hydroxyl, mercapto, lower alkoxy, lower alkyl-thio,halogen, lower alkyl, unsubstituted or substituted mononuclear aryl, or—N(R²)₂;

R¹ is hydrogen or lower alkyl;

each R² is, independently, —R⁷, —(CH₂)_(m)—OR⁸, —(CH₂)_(m)—NR⁷R¹⁰,—(CH₂)_(n)(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸, —(CH₂CH₂O)_(m)—R⁸,—(CH₂CH₂O)_(m)—CH₂CH₂NR⁷R¹⁰, —(CH₂)_(n)—C(═O)NR⁷R¹⁰,—(CH₂)_(n)-(Z)_(g)—R⁷, —(CH₂)_(m)—NR¹⁰—CH₂(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸,—(CH₂)_(n)—CO₂R⁷, or

R³ and R⁴ are each, independently, hydrogen, lower alkyl, hydroxyl-loweralkyl, phenyl, (phenyl)-lower alkyl, (halophenyl)-lower alkyl,((lower-alkyl)phenyl)-lower-alkyl, ((lower-alkoxy)phenyl))-lower alkyl,(naphthyl)-lower alkyl, or (pyridyl)-lower alkyl, or a group representedby formula A or formula B, with the proviso that at least one of R³ andR⁴ is a group represented by the formula A or formula B;

—(C(R^(L))₂)_(o)-x-(C(R^(L))₂)_(p)A¹  formula A:

—(C(R^(L))₂)_(o)-x-(C(R^(L))₂)_(p)A²  formula B:

A¹ is a C₆-C₁₅-membered aromatic carbocycle substituted with at leastone R⁵ and the remaining substituents are R⁶;

A² is a six to fifteen-membered aromatic heterocycle substituted with atleast one R⁵ and the remaining substituents are R⁶ wherein said aromaticheterocycle comprises 1-4 heteroatoms selected from the group consistingof O, N, and S;

each R^(L) is, independently, —R⁷, —(CH₂)_(n)—OR⁸, —O—(CH₂)_(m)—OR⁸,—(CH₂)_(n)—NR⁷R¹⁰, —O—(CH₂)_(m)—NR⁷R¹⁰,—(CH₂)_(n)(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸,—O—(CH₂)_(m)(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸, —(CH₂CH₂O)_(m)—R⁸,—O—(CH₂CH₂O)_(m)—R⁸, —(CH₂CH₂O)_(m)—CH₂CH₂NR⁷R¹⁰,—O—(CH₂CH₂O)_(m)—CH₂CH₂NR⁷R¹⁰, —(CH₂)_(n)—C(O)NR⁷R¹⁰,—O—(CH₂)_(m)—C(═O)NR⁷R¹⁰, —(CH₂)_(n)(Z)_(g)—R⁷, —O—(CH₂)_(m)-(Z)_(g)—R⁷,—(CH₂)_(n)—NR¹⁰—CH₂(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸,—O—(CH₂)_(m)—NR¹⁰—CH₂(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸, —(CH₂)_(n)—CO₂R⁷,—O—(CH₂)_(m)—CO₂R⁷, —OSO₃H, —O-glucuronide, —O-glucose,

each o is, independently, an integer from 0 to 10;

each p is, independently, an integer from 0 to 10;

with the proviso that the sum of o and p in each contiguous chain is

from 1 to 10;

each x is, independently, O, NR¹⁰, C(═O), CHOH, C(═N—R¹⁰), CHNR⁷R¹⁰, ora single bond;

each R⁵ is, independently, -Link-(CH₂)_(m)-CAP,-Link-(CH₂)_(n)(CHOR⁸)(CHOR⁸)_(n)-CAP, -Link-(CH₂CH₂O)_(m)—CH₂-CAP,-Link-(CH₂CH₂O)_(m)—CH₂CH₂-CAP, -Link-(CH₂)_(m)-(Z)_(g)-CAP,-Link-(CH₂)_(n)(Z)_(g)—(CH₂)_(m)-CAP,-Link-(CH₂)_(n)—NR¹³—CH₂(CHOR⁸)(CHOR⁸)_(n)-CAP,-Link-(CH₂)_(n)—(CHOR⁸)_(m)CH₂—NR¹³-(Z)_(g)-CAP,-Link-(CH₂)_(n)NR¹³—(CH₂)_(m)(CHOR⁸)_(n)CH₂NR¹³-(Z)_(g)-CAP,-Link-(CH₂)_(m)-(Z)_(g)—(CH₂)_(m)-CAP, -Link-NH—C(O)—NH(CH₂)_(m)-CAP,-Link-(CH₂)_(m)—C(O)NR¹³—(CH₂)_(m)-CAP,-Link-(CH₂)_(n)-(Z)_(g)—(CH₂)_(m)-(Z)_(g)-CAP, or-Link-Z_(g)-(CH₂)_(m)-Het-(CH₂)_(m)-CAP;

-   -   each R⁶ is, independently, hydrogen, lower alkyl, phenyl,        substituted phenyl o-CH₂(CHOR⁸)_(m)—CH₂OR⁸, —OR¹¹, —N(R⁷)₂,        —(CH₂)_(m)—OR⁸, —O—(CH₂)_(m)—OR⁸, —(CH₂)_(n)—NR⁷R¹⁰,        —O—(CH₂)_(m)—NR⁷R¹⁰, —(CH₂)_(n)(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸,        O—(CH₂)_(m)(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸, —(CH₂CH₂O)_(m)—R⁸,        —O—(CH₂CH₂O)_(m)R⁸, —(CH₂CH₂O)_(m)—CH₂CH₂NR⁷R¹⁰,        —O—(CH₂CH₂O)_(m)CH₂CH₂NR⁷R¹⁰, —(CH₂)_(n)—C(O)NR⁷R¹⁰,        —O—(CH₂)_(m)—C(O)NR⁷R¹⁰, —(CH₂)_(n)—(Z)_(g)—R⁷,        —O—(CH₂)_(m)-(Z)_(g)—R⁷,        —(CH₂)_(n)—NR¹⁰—CH₂(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸,        —O—(CH₂)_(m)—NR¹⁰—CH₂(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸,        —(CH₂)_(n)—CO₂R⁷, —O—(CH₂)_(m)—CO₂R⁷, —OSO₃H, —O-glucuronide,        —O-glucose,

wherein when two R⁶ are —OR¹¹ and are located adjacent to each other onthe aromatic carbocycle or aromatic heterocycle, the two OR¹¹ may form amethylenedioxy group;

each R⁷ is, independently, hydrogen, lower alkyl, phenyl, substitutedphenyl or —CH₂(CHOR⁸)_(m)—CH₂OR⁸;

each R⁸ is, independently, hydrogen, lower alkyl, —C(═O)—R¹¹,glucuronide, 2-tetrahydropyranyl, or

each R⁹ is, independently, —CO₂R⁷, —CON(R⁷)₂, —SO₂CH₃, —C(═O)R⁷,—CO₂R¹³, —CON(R¹³)₂, —SO₂CH₂R¹³, or —C(═O)¹³;

each R¹⁰ is, independently, —H, —SO₂CH₃, —CO₂R, —C(═O)NR⁷R⁹, —C(═O)R⁷,or —CH₂—(CHOH)_(n)—CH₂OH;

each Z is, independently, —(CHOH)—, —C(═O)—, —(CHNR⁷R¹⁰)—, —(C═NR¹⁰)—,—NR¹⁰—, —(CH₂)_(n)—, —(CHNR¹³R¹³)—, —(C═NR¹³)—, or —NR¹³—;

each R¹¹ is, independently, hydrogen, lower alkyl, phenyl lower alkyl orsubstituted phenyl lower alkyl;

each R¹² is, independently, —SO₂CH₃, —CO₂R⁷, —C(═O)NR⁷R⁹, —C(═O)R⁷,—CH₂(CHOH)_(n)—CH₂OH, —CO₂R⁷, —C(═O)NR⁷R⁷, or —C(═O)R⁷;

each R¹³ is, independently, hydrogen, lower alkoxy, R¹⁰, R¹¹, R¹², —OR⁷,—(CH₂)_(m)—NR⁷R¹⁰, —(CH₂)_(m)—NR⁷R⁷, —(CH₂)_(m)—NR¹¹R¹¹,—(CH₂)_(m)—(NR¹¹R¹¹R¹¹)⁺, —(CH₂)_(m)—(CHOR⁸)_(m)—(CH₂)_(m)NR¹¹R¹¹,—(CH₂)_(m)—(CHOR⁸)_(m)—(CH₂)_(m)NR⁷R¹⁰, —(CH₂)_(m)—NR¹⁰R¹⁰,—(CH₂)_(m)—(CHOR⁸)_(m)—(CH₂)_(m)—(NR¹¹R¹¹R¹¹)⁺,—(CH₂)_(m)—(CHOR⁸)_(m)—(CH₂)_(m)NR⁷R⁷,

with the proviso that in the moiety —NR¹³R¹³, the two R¹³ along with thenitrogen to which they are attached may, optionally, form a ringselected from:

each V is, independently, —(CH₂)_(m)—NR⁷R¹⁰, —(CH₂)_(m)—NR⁷R⁷,—(CH₂)_(m)—(NR¹¹R¹¹R¹¹)⁺, —(CH₂)_(n)—(CHOR⁸)_(m)—(CH₂)_(m)NR⁷R¹⁰,—(CH₂)_(n)—NR¹⁰R¹⁰—(CH₂)_(n)—(CHOR⁸)_(m)—(CH₂)_(m)NR⁷R⁷,—(CH₂)_(n)—(CHOR⁸)_(m)—(CH₂)_(m), —(NR¹¹R¹¹R¹¹)⁺ with the proviso thatwhen V is attached directly to a nitrogen atom, then V can also be,independently, R⁷, R¹⁰, or (R¹¹)₂;

each R¹⁴ is, independently, H, R¹², —(CH₂)_(n)—SO₂CH₃,—(CH₂)_(n)—CO₂R¹³, —(CH₂)_(n)—C(═O)NR¹³R¹³, —(CH₂)_(n)—C(═O)R¹³,—(CH₂)_(n)—(CHOH)_(n)—CH₂OH, —NH—(CH₂)_(n)—SO₂CH₃,NH—(CH₂)_(n)—C(═O)R¹¹, NH—C(═O)—NH—C(═O)R¹¹, —C(═O)NR¹³R¹³, —OR¹¹,—NH—(CH₂)_(n)—R¹⁰, —Br, —Cl, —F, —I, SO₂NHR¹¹, —NHR¹³, —NH—C(═O)—NR³R¹³,—(CH₂)_(n)—NHR¹³, or —NH—(CH₂)_(n)—C(═O)—R¹³;

each g is, independently, an integer from 1 to 6;

each m is, independently, an integer from 1 to 7;

each n is, independently, an integer from 0 to 7;

each -Het- is, independently, —N(R⁷)—, —N(R¹⁰)—, —S—, —SO—, —SO₂—; —O—,—SO₂NH—, —NHSO₂—, —NR⁷CO—, —CONR⁷—, —N(R¹³)—, —SO₂NR¹³—, —NR¹³CO—, or—CONR₁₃—;

each Link is, independently, —O—, —(CH₂)_(n)—, —O(CH₂)_(m)—,—NR¹³—C(═O)—NR¹³—, —NR¹³—C(═O)—(CH₂)_(m)—, —C(═O)NR¹³—(CH₂)_(m) ⁻,—(CH₂)_(n)-(Z)_(g)—(CH₂)_(n)—, —S—, —SO—, —SO₂—, —SO₂NR⁷—, —SO₂NR¹⁰—, or-Het-;

each CAP is, independently

with the proviso that when any —CHOR⁸— or —CH₂OR⁸ groups are located1,2- or 1,3- with respect to each other, the R⁸ groups may, optionally,be taken together to form a cyclic mono- or di-substituted 1,3-dioxaneor 1,3-dioxolane.

The present also provides pharmaceutical compositions which comprise acompound described herein.

The present invention also provides a method of promoting hydration ofmucosal surfaces, comprising:

administering an effective amount of a compound described herein to amucosal surface of a subject.

The present invention also provides a method of restoring mucosaldefense, comprising:

topically administering an effective amount of compound described hereinto a mucosal surface of a subject in need thereof.

The present invention also provides a method of blocking ENaC,comprising:

contacting sodium channels with an effective amount of a compoundrepresented by described herein.

The present invention also provides a method of promoting mucusclearance in mucosal surfaces, comprising:

administering an effective amount of a compound represented describedherein to a mucosal surface of a subject.

The present invention also provides a method of treating dry eye,comprising:

administering an effective amount of a compound described herein to theeye of the subject in need thereof.

The present invention also provides a method of treating Sjogren'sdisease-associated dry eye, comprising:

administering an effective amount of a compound described herein to theeye of the subject in need thereof.

The present invention also provides a method of treating eyeinflammation caused by dry eye, comprising:

administering an effective amount of a compound described herein to theeye of the subject in need thereof.

The present invention also provides a method of promoting ocularhydration, comprising:

administering an effective amount of a compound described herein to theeye of the subject.

The present invention also provides a method of promoting cornealhydration, comprising:

administering an effective amount of a compound described herein to theeye of the subject.

The present invention also provides a method of treating chronicbronchitis, comprising:

administering an effective amount of a compound described herein to asubject in need thereof.

The present invention also provides a method of treating cysticfibrosis, comprising:

administering an effective amount of compound described herein to asubject in need thereof.

The present invention also provides a method of treating rhinosinusitis,comprising:

administering an effective amount of a compound described herein to asubject in need thereof.

The present invention also provides a method of treating nasaldehydration, comprising:

administering an effective amount of a compound described herein to thenasal passages of a subject in need thereof.

In a specific embodiment, the nasal dehydration is brought on byadministering dry oxygen to the subject.

The present invention also provides a method of treating sinusitis,comprising:

administering an effective amount of a compound described herein to asubject in need thereof.

The present invention also provides a method of treating pneumonia,comprising:

administering an effective amount of a compound described herein to asubject in need thereof.

The present invention also provides a method of treatingventilator-induced pneumonia, comprising:

administering an effective compound described herein to a subject bymeans of a ventilator.

The present invention also provides a method of treating asthma,comprising:

administering an effective amount of a compound described herein to asubject in need thereof.

The present invention also provides a method of treating primary ciliarydyskinesia, comprising:

administering an effective amount of a compound described herein to asubject in need thereof.

The present invention also provides a method of treating otitis media,comprising:

administering an effective amount of a compound described herein to asubject in need thereof.

The present invention also provides a method of inducing sputum fordiagnostic purposes, comprising:

administering an effective amount of compound described herein to asubject in need thereof.

The present invention also provides a method of treating chronicobstructive pulmonary disease, comprising:

administering an effective amount of a compound described herein to asubject in need thereof.

The present invention also provides a method of treating emphysema,comprising:

administering an effective amount of a compound described herein to asubject in need thereof.

The present invention also provides a method of treating Sjögren'sdisease, comprising:

administering an effective amount of compound described herein to asubject in need thereof.

The present invention also provides a method of treating vaginaldryness, comprising:

administering an effective amount of a compound described herein to thevaginal tract of a subject in need thereof

The present invention also provides a method of treating dry skin,comprising:

administering an effective amount of a compound described herein to theskin of a subject in need thereof.

The present invention also provides a method of treating dry mouth(xerostomia), comprising:

administering an effective amount of compound described herein to themouth of the subject in need thereof.

The present invention also provides a method of treating distalintestinal obstruction syndrome, comprising:

administering an effective amount of compound described herein to asubject in need thereof.

The present invention also provides a method of treating esophagitis,comprising:

administering an effective amount of a compound described herein to asubject in need thereof.

The present invention also provides a method of treating bronchiectasis,comprising:

administering an effective amount of a compound described herein to asubject in need thereof.

The present invention also provides a method of treating constipation,comprising:

administering an effective amount of a compound described herein to asubject in need thereof. In one embodiment of this method, the compoundis administered either orally or via a suppository or enema.

The present invention also provides a method of treating chronicdiverticulitis comprising:

administering an effective amount of a compound described herein to asubject in need thereof.

It is an object of the present invention to provide treatmentscomprising the use of osmolytes together with sodium channel blockers offormula (I) that are more potent, more specific, and/or absorbed lessrapidly from mucosal surfaces, and/or are less reversible as compared tocompounds such as amiloride, benzamil, and phenamil.

It is another aspect of the present invention to provide treatmentsusing sodium channel blockers of formula (I) that are more potent and/orabsorbed less rapidly and/or exhibit less reversibility, as compared tocompounds such as amiloride, benzamil, and phenamil when administeredwith an osmotic enhancer. Therefore, such sodium channel blockers whenused in conjunction with osmolytes will give a prolonged pharmacodynamichalf-life on mucosal surfaces as compared to either compound used alone.

It is another object of the present invention to provide treatmentsusing sodium channel blockers of formula (I) and osmolytes togetherwhich are absorbed less rapidly from mucosal surfaces, especially airwaysurfaces, as compared to compounds such as amiloride, benzamil, andphenamil.

It is another object of the invention to provide compositions whichcontain sodium channel blockers of formula (I) and osmolytes.

The objects of the invention may be accomplished with a method oftreating a disease ameliorated by increased mucociliary clearance andmucosal hydration comprising administering an effective amount of acompound of formula (I) as defined herein and an osmolyte to a subjectto a subject in need of increased mucociliary clearance and mucosalhydration.

The objects of the invention may also be accomplished with a method ofinducing sputum for diagnostic purposes, comprising administering aneffective amount of a compound of formula (I) as defined herein and anosmolyte to a subject in need thereof.

The objects of the invention may also be accomplished with a method oftreating anthrax, comprising administering an effective amount of acompound of formula (I) as defined herein and an osmolyte to a subjectin need thereof.

The objects of the invention may also be accomplished with a method ofprophylactic, post-exposure prophylactic, preventive or therapeutictreatment against diseases or conditions caused by pathogens,particularly pathogens which may be used in bioterrorism, comprisingadministering an effective amount of a compound of formula (I) to asubject in need thereof.

The objects of the invention may also be accomplished with acomposition, comprising a compound of formula (I) as defined herein andan osmolyte as defined herein.

BRIEF DESCRIPTION OF THE DRAWINGS

A more complete appreciation of the invention and many of the advantagesthereof may be readily obtained by reference to the information hereinin conjunction with the following figures:

FIG. 1: Tear volume assessments over 6 hours in ExLac rats withAmiloride. Tear volume from ExLac and Normal rats treated with a vehicleare shown for comparison. Error bars are standard error.

FIG. 2: Tear volume assessments over 6 hours in ExLac rats with Compound51. Tear volume from ExLac and Normal rats treated with a vehicle areshown for comparison. Error bars are standard error.

FIG. 3: Tear volume assessments over 6 hours in ExLac rats with Compound75. Tear volume from ExLac and Normal rats treated with a vehicle areshown for comparison. Error bars are standard error.

FIG. 4: Tear volume assessments over 6 hours in ExLac rats with CompoundP-59. Tear volume from ExLac and Normal rats treated with a vehicle areshown for comparison. Error bars are standard error.

FIG. 5: Tear volume assessments over 6 hours in ExLac rats with Compound46. Tear volume from ExLac and Normal rats treated with a vehicle areshown for comparison. Error bars are standard error.

FIG. 6: Tear volume assessments over 6 hours in ExLac rats with Compound45. Tear volume from ExLac and Normal rats treated with a vehicle areshown for comparison. Error bars are standard error.

FIG. 7: Tear volume assessments over 6 hours in ExLac rats with Compound145. Tear volume from ExLac and Normal rats treated with a vehicle areshown for comparison. Error bars are standard error.

FIG. 8: Tear volume assessments over 6 hours in ExLac rats with Compound82. Tear volume from ExLac and Normal rats treated with a vehicle areshown for comparison. Error bars are standard error.

FIG. 9: Tear volume assessments over 6 hours in ExLac rats with Compound15. Tear volume from ExLac and Normal rats treated with a vehicle areshown for comparison. Error bars are standard error.

FIG. 10: Tear volume assessments over 6 hours in ExLac rats withCompound 9. Tear volume from ExLac and Normal rats treated with avehicle are shown for comparison. Error bars are standard error.

FIG. 11: Tear volume assessments over 6 hours in ExLac rats withCompound 42. Tear volume from ExLac and Normal rats treated with avehicle are shown for comparison. Error bars are standard error.

FIG. 12: Tear volume assessments over 6 hours in ExLac rats withCompound 116. Tear volume from ExLac and Normal rats treated with avehicle are shown for comparison. Error bars are standard error.

FIG. 13: Tear volume assessments over 6 hours in ExLac rats withCompound 102. Tear volume from ExLac and Normal rats treated with avehicle are shown for comparison. Error bars are standard error.

FIG. 14: Tear volume assessments over 6 hours in ExLac rats withCompound 133. Tear volume from ExLac and Normal rats treated with avehicle are shown for comparison. Error bars are standard error.

FIG. 15: Tear volume assessments over 6 hours in ExLac rats withCompound 90. Tear volume from ExLac and Normal rats treated with avehicle are shown for comparison. Error bars are standard error.

FIG. 16: Confocal images showing the x-z reconstruction of mouse corneasimaged as either the corneal cells (Calcein labeled) or the treatmentdrug (amiloride or Compound 9) taken one hour after application to thecorneal epithelium.

DETAILED DESCRIPTION OF THE INVENTION

As used herein, the following terms are defined as indicated.

“A compound of the invention” means a compound of Formula I or a salt,particularly a pharmaceutically acceptable salt thereof.

“A compound of Formula I” means a compound having the structural formuladesignated herein as Formula I. Compounds of Formula I include solvatesand hydrates (i.e., adducts of a compound of Formula I with a solvent).In those embodiments wherein a compound of Formula I includes one ormore chiral centers, the phrase is intended to encompass each individualstereoisomer including optical isomers (enantiomers and diastereomers)and geometric isomers (cis-/trans-isomerism) and mixtures ofstereoisomers. In addition, compounds of Formula I also includetautomers of the depicted formula(s).

Throughout the description and examples, compounds are named usingstandard IUPAC naming principles, where possible, including the use ofthe ChemDraw Ultra 11.0 software program for naming compounds, sold byCambridgeSoft Corp./PerkinElmer.

In some chemical structure representations where carbon atoms do nothave a sufficient number of attached variables depicted to produce avalence of four, the remaining carbon substituents needed to provide avalence of four should be assumed to be hydrogen. Similarly, in somechemical structures where a bond is drawn without specifying theterminal group, such bond is indicative of a methyl (Me, —CH₃) group, asis conventional in the art.

The present invention is based on the discovery that the compounds offormula (I) are more potent and/or absorbed less rapidly from mucosalsurfaces, and/or are less reversible as compared to known compounds.

The present invention is also based on the discovery that the compoundsof formula (I) are more potent and/or absorbed less rapidly and/orexhibit less reversibility, as compared to compounds such as amilorde,benzamil, and phenamil. Therefore, the compounds will give a prolongedpharmacodynamic half-life on mucosal surfaces as compared to knowncompounds.

The present invention is also based on the discovery that certaincompounds embraced by formula (I) (1) are absorbed less rapidly frommucosal surfaces, especially ocular surfaces, as compared to knowncompounds and (2) when absorbed from musosal surfaces afteradministration to the mucosal surfaces, are excreated mainly non-renallyin order to minimize the chances of hyperkalemia. The present inventionis also based on the discovery that certain compounds embraced byformula (I) (1) are absorbed less rapidly from mucosal surfaces,especially ocular surfaces, as compared to known compounds and (2) areconverted in vivo into metabolic derivitives thereof which have reducedefficacy in blocking sodium channels as compared to the administeredparent compound in order to minimize the chances of hyperkalemia.

The present invention is also based on the discovery that certaincompounds embraced by formula (I) (1) are absorbed less rapidly frommucosal surfaces, especially ocular surfaces, as compared to knowncompounds and (2) are not converted in vivo into metabolic derivativesthereof which have enhanced or similar efficacy in blocking sodiumchannels as compared to the administered parent compound in order tominimize the chances of hyperkalemia

The present invention is also based on the discovery that certaincompounds embraced by formula (I) provide methods of treatment that takeadvantage of the pharmacological properties of the compounds describedabove.

In particular, the present invention is also based on the discovery thatcertain compounds embraced by formula (I) rehydrate mucosal surfaces.

In particular, the present invention is also based on the discovery thatcertain compounds embraced by formula (I) are useful in treating dry eyeand related ocular diseases.

In the compounds represented by formula (I), X may be hydrogen, halogen,trifluoromethyl, lower alkyl, lower cycloalkyl, unsubstituted orsubstituted phenyl, lower alkyl-thio, phenyl-lower alkyl-thio, loweralkyl-sulfonyl, or phenyl-lower alkyl-sulfonyl. Halogen is preferred.

Examples of halogen include fluorine, chlorine, bromine, and iodine.Chlorine and bromine are the preferred halogens. Chlorine isparticularly preferred. This description is applicable to the term“halogen” as used throughout the present disclosure.

As used herein, the term “lower alkyl” means an alkyl group having lessthan 8 carbon atoms. This range includes all specific values of carbonatoms and subranges there between, such as 1, 2, 3, 4, 5, 6, and 7carbon atoms. The term “alkyl” embraces all types of such groups, e.g.,linear, branched, and cyclic alkyl groups. This description isapplicable to the term “lower alkyl” as used throughout the presentdisclosure. Examples of suitable lower alkyl groups include methyl,ethyl, propyl, cyclopropyl, butyl, isobutyl, etc.

Substituents for the phenyl group include halogens. Particularlypreferred halogen substituents are chlorine and bromine.

Y may be hydrogen, hydroxyl, mercapto, lower alkoxy, lower alkyl-thio,halogen, lower alkyl, lower cycloalkyl, mononuclear aryl, or —N(R²)₂.The alkyl moiety of the lower alkoxy groups is the same as describedabove. Examples of mononuclear aryl include phenyl groups. The phenylgroup may be unsubstituted or substituted as described above. Thepreferred identity of Y is —N(R²)₂. Particularly preferred are suchcompounds where each R² is hydrogen.

R¹ may be hydrogen or lower alkyl. Hydrogen is preferred for R¹.

Each R² may be, independently, —R⁷, —(CH₂)_(m)—OR⁸, —(CH₂)_(m)—NR⁷R¹⁰,—(CH₂)_(n)(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸, —(CH₂CH₂O)_(m)—R⁸,—(CH₂CH₂O)_(m)—CH₂CH₂NR⁷R¹⁰, —(CH₂)_(n)—C(═O)NR⁷R¹⁰,—(CH₂)_(n)-(Z)_(g)—R⁷, —(CH₂)_(m)—NR¹⁰—CH₂(CHOR⁸)(CHOR₈)_(n)—CH₂OR⁸,—(CH₂)_(n)—CO₂R⁷, or

Hydrogen and lower alkyl, particularly C₁-C₃ alkyl, are preferred forR². Hydrogen is particularly preferred.

R³ and R⁴ may be, independently, hydrogen, lower alkyl, hydroxyl-loweralkyl, phenyl, (phenyl)-lower alkyl, (halophenyl)-lower alkyl,((lower-alkyl)phenyl)-lower-alkyl), (lower-alkoxyphenyl)-lower alkyl,(naphthyl)-lower alkyl, (pyridyl)-lower alkyl or a group represented by—(C(R^(L))₂)_(o)—(C(R^(L))₂)_(p)A¹ or—(C(R^(L))₂)_(o)-x-(C(R^(L))₂)_(p)A², provided that at least one of R³and R⁴ is a group represented by —(C(R^(L))₂)_(o)-x-(C(R^(L))₂)_(p)A¹ or—(C(R^(L))₂)_(o)-x-(C(R^(L))₂)_(p)A².

Preferred compounds are those where one of R³ and R⁴ is hydrogen and theother is represented by —(C(R^(L))₂)_(o)-x-(C(R^(L))₂)_(p)A¹ or—(C(R^(L))_(o)-x-(C(R^(L))₂)_(p)A². In a particularly preferred aspectone of R³ and R⁴ is hydrogen and the other of R³ or R⁴ is represented by—(C(R^(L))₂)_(o)-x-(C(R^(L))₂)_(p)A¹. In another particularly preferredaspect one of R³ and R is hydrogen and the other of R³ or R⁴ isrepresented by —(C(R^(L))₂)_(o)-x-(C(R^(L))₂)_(p)A².

A moiety —(C(R^(L))₂)_(o)-x-(C(R^(L))₂)_(p)— defines an alkylene groupbonded to the group A¹ or A². The variables o and p may each,independently, be an integer from 0 to 10, subject to the proviso thatthe sum of o and p in the chain is from 1 to 10. Thus, o and p may eachbe 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. Preferably, the sum of o and pis from 2 to 6. In a particularly preferred embodiment, the sum of o andp is 4.

The linking group in the alkylene chain, x, may be, independently, O,NR¹⁰, C(═O), CHOH, C(═N—R¹⁰), CHNR⁷R¹⁰, or a single bond:

Therefore, when x is a single bond, the alkylene chain bonded to thering is represented by the formula —(C(R^(L))₂)_(o+p)—, in which the sumo+p is from 1 to 10.

Each R^(L) may be, independently, —R⁷, —(CH₂)_(n)—OR⁸, —O—(CH₂)_(m)—OR⁸,—(CH₂)_(n)—NR⁷R¹⁰, —O—(CH₂)_(m)—NR⁷R¹⁰,—(CH₂)_(n)(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸,—O—(CH₂)_(m)(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸, —(CH₂CH₂O)_(m)—R⁸,—O—(CH₂CH₂O)_(m)—R⁸, —(CH₂CH₂O)_(m)—CH₂CH₂NR⁷R¹⁰,—O—(CH₂CH₂O)_(m)—CH₂CH₂NR⁷R¹⁰, —(CH₂)_(n)—C(═O)NR⁷R¹⁰,—O—(CH₂)_(m)—C(═O)NR⁷R¹⁰, —(CH₂)_(n)—(Z)_(g)—R⁷,—O—(CH₂)_(m)-(Z)_(g)—R⁷, —(CH₂)_(n)—NR¹⁰—CH₂(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸,—O—(CH₂)_(m)—NR¹⁰—CH₂(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸, —(CH₂)_(n)—CO₂R⁷,—O—(CH₂)_(m)—CO₂R⁷, —OSO₃H, —O-glucuronide, —O-glucose,

The term —O-glucuronide, unless otherwise specified, means a grouprepresented by

wherein the

means the glycosidic linkage can be above or below the plane of thering.

The term —O-glucose, unless otherwise specified, means a grouprepresented by

wherein the

means the glycosidic linkage can be above or below the plane of thering.

The preferred R^(L) groups include —H, —OH, —N(R⁷)₂, especially whereeach R⁷ is hydrogen.

In the alkylene chain in —(C(R^(L))₂)_(o)-x-(C(R^(L))₂)_(p)A or—(C(R^(L))₂)_(o)-x-(C(R^(L))₂)_(p)A², it is preferred that when oneR^(L) group bonded to a carbon atoms is other than hydrogen, then theother R^(L) bonded to that carbon atom is hydrogen, i.e., the formula—CHR^(L)—. It is also preferred that at most two R^(L) groups in analkylene chain are other than hydrogen, wherein the other R^(L) groupsin the chain are hydrogens. Even more preferably, only one R^(L) groupin an alkylene chain is other than hydrogen, wherein the other R^(L)groups in the chain are hydrogens. In these embodiments, it ispreferable that x is a single bond.

In another particular embodiment of the invention, all of the R^(L)groups in the alkylene chain are hydrogen. In these embodiments, thealkylene chain is represented by the formula —(CH₂)_(o)-x-(CH₂)_(p)—.

A¹ is a C₆-C₁₅-membered aromatic carbocycle substituted with at leastone R⁵ and the remaining substituents are R⁶. The term aromatic is wellknown term of chemical art and designates conjugated systems of 4n′+2electrons that are within a ring system, that is with 6, 10, 14, etc.π-electrons wherein, according to the rule of Huckel, n′ is 1, 2, 3,etc. The 4n′+2 electrons may be in any size ring including those withpartial saturation so long as the electrons are conjugated. Forinstance, but not by way of limitation, 5H-cyclohepta-1,3,5-triene,benzene, naphthalene, 1,2,3,4-tetrahydronaphthalene etc. would all beconsidered aromatic.

The C₆-C₁₅ aromatic carbocycle may be monocyclic, bicyclic, or tricyclicand may include partially saturated rings. Non-limiting examples ofthese aromatic carbocycles comprise benzene, 5H-cyclohepta-1,3,5-triene,naphthalene, phenanthrene, azulene, anthracene,1,2,3,4-tetrahydronapthalene, 1,2-dihydronapthalene, indene,5H-dibenzo[a,d]cycloheptene, etc.

The C₆-C₁₅ aromatic carbocycle may be attached to the—(C(R^(L))₂)_(o)-x-(C(R^(L))₂)_(p)-moiety through any ring carbon atomas appropriate, unless otherwise specified. Therefore, when partiallysaturated bicyclic aromatic is 1,2-dihydronapthalene, it may be1,2-dihydronapthalen-1-yl, 1,2-dihydronapthalen-3-yl,1,2-dihydronapthalen-5-yl, etc. In a preferred embodiment A¹ is phenyl,indenyl, napthalenyl, 1,2-dihydronapthalenyl,1,2,3,4-tetrahydronapthalenyl, anthracenyl, fluorenyl, phenanthrenyl,azulenyl, cyclohepta-1,3,5-trienyl or 5H-dibenzo[a,d]cycloheptenyl. Inanother preferred embodiment, A¹ is napthalen-1-yl. In another preferredembodiment, A¹ is napthalen-2-yl.

In another preferred embodiment, A¹ is

wherein each Q is, independently, C—H, C—R⁵, or C—R⁶, with the provisothat at least one Q is C—R⁵. Therefore, Q may be 1, 2, 3, 4, 5, or 6C—H. Therefore, Q may be 1, 2, 3, 4, 5, or 6 C—R⁶. In a particularlypreferred embodiment, each R⁶ is H.

In another preferred embodiment, A¹ is

wherein each Q is, independently, C—H, C—R⁵, C—R⁶, with the proviso thatat least one Q is C—R⁵. Therefore, Q may be 1, 2, 3, 4, 5, or 6 C—H.Therefore, Q may be 1, 2, 3, 4, 5, or 6 C—R⁶. In a particularlypreferred embodiment, each R⁶ is H.

In another preferred embodiment, A¹ is

wherein each Q is, independently, C—H, C—R⁵, or C—R⁶, with the provisothat at least one Q is C—R⁵. Therefore, Q may be 1, 2, 3, or 4 C—H.Therefore, Q may be 1, 2, 3, or 4 C—R⁶. In a particularly preferredembodiment, each R⁶ is H.

In a particularly preferred embodiment, A¹ is

In another particularly preferred embodiment, A¹ is

In another particularly preferred embodiment, A¹ is

A² is a six to fifteen-membered aromatic heterocycle substituted with atleast one R⁵ and the remaining substituents are R⁶ wherein the aromaticheterocycle comprises 1-4 heteroatoms selected from the group consistingof O, N, and S.

The six to fifteen-membered aromatic heterocycle may be monocyclic,bicyclic, or tricyclic and may include partially saturated rings. Nonlimiting examples of these aromatic heterocycles include pyridine,pyrazine, triazine, 1H-azepine, benzo[b]furan, benzo[b]thiophene,isobenzofuran, isobenzothiophene, 2,3-dihydrobenzo[b]furan,benzo[b]thiophene, 2,3-diydrobenzo[b]thiophene, indolizine, indole,isoindole benzoxazole, benzimidazole, indazole, benzisoxazole,benzisothizole, benzopyrazole, benzoxadiazole, benzothiadiazole,benzotriazole, purine, quinoline, 1,2,3,4-tetrahydroquinoline,3,4-dihydro-2H-chromene, 3,4-dihydro-2H-thiochromene, isoquinoline,cinnoline, quinolizine, phthalazine, quinoxaline, quinazoline,naphthiridine, pteridine, benzopyrane, pyrrolopyridine, pyrrolopyrazine,imidazopyrdine, pyrrolopyrazine, thienopyrazine, furopyrazine,isothiazolopyrazine, thiazolopyrazine, isoxazolopyrazine,oxazolopyrazine, pyrazolopyrazine, imidazopyrazine, pyrrolopyrimidine,thienopyrimidine, furopyrimidine, isothiazolopyrimidine,thiazolopyrimidine, isoxazolopyrimidine, oxazolopyrimidine,pyrazolopyrimidine, imidazopyrimidine, pyrrolopyridazine,thienopyridazine, furopyridazine, isothiazolopyridazine,thiazolopyridazine, oxazolopyridazine, thiadiazolopyrazine,oxadiazolopyrimidine, thiadiazolopyrimidine, oxadiazolopyridazine,thiazolopyridazine, imidazooxazole, imidazothiazole, imidazoimidazole,isoxazolotriazine, isothiazolotriazine, oxazolotriazine,thiazolotriazine, carbazole, acridine, phenazine, phenothiazine,phenooxazine, and 5H-dibenz[b,f]azepine,10,11-dihydro-5H-dibenz[b,f]azepine, etc.

The six to fifteen-membered aromatic heterocycle may be attached to the—(C(R^(L))₂)_(o)-x-(C(R^(L))₂)_(p)— moiety through any ring carbon atomor ring nitrogen atom so long as a quanternary nitrogen atom is notformed by the attachment. Therefore, when partially saturated aromaticheterocycle is 1H-azepine, it may be 1H-azepin-1-yl, 1H-azepin-2-yl,1H-azepin-3-yl, etc. Preferred aromatic heterocycles are indolizinyl,indolyl, isoindolyl, indolinyl, benzo[b]furanyl,2,3-dihydrobenzo[b]furanyl, benzo[b]thiophenyl,2,3-diydrobenzo[b]thiophenyl, indazolyl, benzimidazolyl, benzthiazolyl,purinyl, quinolinyl, 1,2,3,4-tetrahydroquinolinyl,3,4-dihydro-2H-chromenyl, 3,4-dihydro-2H-thiochromenyl, isoquinolinyl,cinnolinyl, phthalazinyl, quinazolinyl, quinoxalinyl,1,8-naphthyridinyl, pteridinyl, carbazolyl, acridinyl, phenazinyl,phenothiazinyl, phenoxazinyl, dibenzofuranyl, dibenzothiophenyl,1H-azepinyl, 5H-dibenz[b,f]azepinyl, are10,11-dihydro-5H-dibenz[b,f]azepinyl.

In another preferred embodiment, A² is

wherein each Q is, independently, C—H, C—R⁵, C—R⁶, or a nitrogen atom,with the proviso that at least one Q is nitrogen and one Q is C—R⁵, andat most three Q in a ring are nitrogen atoms. Therefore, in any onering, each Q may be 1, 2, or 3 nitrogen atoms. In a preferredembodiment, only one Q in each ring is nitrogen. In another preferredembodiment, only a single Q is nitrogen. Optionally, 1, 2, 3, 4, or 5 Qmay be C—R⁶. Optionally, Q may be 1, 2, 3, 4, or 5 C—H. In aparticularly preferred embodiment, each R⁶ is H.

In another preferred embodiment, A² is

wherein each Q is, independently, C—H, C—R⁵, C—R⁶, or a nitrogen atom,with the proviso that at least one Q is nitrogen and one Q is C—R⁵, andat most three Q in a ring are nitrogen atoms. Therefore, in any onering, each Q may be 1, 2, or 3 nitrogen atoms. In a preferredembodiment, only one Q in each ring is nitrogen. In another preferredembodiment, only a single Q is nitrogen. Optionally, Q may be 1, 2, 3,4, or 5 C—H. Optionally, 1, 2, 3, 4, or 5 Q may be C—R⁶. In aparticularly preferred embodiment, each R⁶ is H.

In another preferred embodiment, A² is

wherein each Q is, independently, C—H, C—R⁵, C—R⁶, or a nitrogen atom,with the proviso that at least one Q is nitrogen and one Q is C—R⁵, andat most three Q in a ring are nitrogen atoms. Therefore, each Q may be1, 2, or 3 nitrogen atoms. In a preferred embodiment, only one Q in eachring is nitrogen. In another preferred embodiment, only a single Q isnitrogen. Optionally, 1, 2, or 3, Q may be C—R⁶. Optionally, Q may be 1,2, or 3 C—H. In a particularly preferred embodiment, each R⁶ is H.

Each R⁵ is, independently, -Link-(CH₂)_(m)-CAP,-Link-(CH₂)_(n)(CHOR⁸)(CHOR⁸)_(n)-CAP, -Link-(CH₂CH₂O)_(m)—CH₂-CAP,-Link-(CH₂CH₂O)_(m)—CH₂CH₂-CAP, -Link-(CH₂)_(m)-(Z)_(g)-CAP,-Link-(CH₂)_(n)(Z)_(g)—(CH₂)_(m)-CAP,-Link-(CH₂)_(n)—NR¹³—CH₂(CHOR⁸)(CHOR⁸)_(n)-CAP,-Link-(CH₂)_(n)—(CHOR⁸)_(m)CH₂—NR¹³-(Z)_(g)-CAP,-Link-(CH₂)_(n)NR¹³—(CH₂)_(m)(CHOR⁸)_(n)CH₂NR¹³-(Z)_(g)-CAP,-Link-(CH₂)_(m)-(Z)_(g)—(CH₂)_(m)CAP, -Link-NH—C(O)—NH—(CH₂)_(m)-CAP,-Link-(CH₂)_(m)C(═O)NR¹³—(CH₂)_(m)-CAP,-Link-(CH₂)_(n)(Z)_(g)—(CH₂)_(m)-(Z)_(g)-CAP, or-Link-Z_(g)-(CH₂)_(m)-Het-(CH₂)_(m)-CAP;

In a preferred embodiment, R⁵ is -Link-(CH₂)_(m)-CAP.

In another preferred embodiment R⁵ is one of the following:

-Link-(CH₂)_(n)(CHOR⁸)(CHOR⁸)_(n)-CAP, -Link-(CH₂CH₂O)_(m)—CH₂-CAP,-Link-(CH₂CH₂O)_(m)—CH₂CH₂-CAP, -Link-(CH₂)_(m)-(Z)_(g)-CAP,-Link-(CH₂)_(n)(Z)_(g)—(CH₂)_(m)-CAP

In another preferred embodiment R⁵ is one of the following:-Link-(CH₂)_(n)—NR¹³—CH₂(CHOR⁸)(CHOR⁸)_(n)-CAP,-Link-(CH₂)_(n)—(CHOR⁸)_(m)CH₂—NR¹³-(Z)_(g)-CAP.-Link-(CH₂)_(n)NR¹³—(CH₂)_(m)(CHOR⁸)_(n)CH₂NR¹³-(Z)_(g)-CAP,-Link(CH₂)_(m)-(Z)_(g)—(CH₂)_(m)CAP, -Link-NH—C(═O)—NH—(CH₂)_(m)-CAP,-Link-(CH₂)_(m)—C(═O)NR¹³—(CH₂)_(m)-CAP,-Link-(CH₂)_(n)-(Z)_(g)—(CH₂)_(m)-(Z)_(g)-CAP, or-Link-Z_(g)-(CH₂)_(m)-Het-(CH₂)_(m)-CAP;

In a particularly preferred embodiment, R⁵ is

Selected substituents within the compounds of the invention are presentto a recursive degree. In this context, “recursive substituent” meansthat a substituent may recite another instance of itself. Because of therecursive nature of such substituents, theoretically, a large number ofcompounds may be present in any given embodiment. For example, R⁹contains a R¹³ substituent. R¹³ can contain an R¹⁰ substituent and R¹⁰can contain a R⁹ substituent. One of ordinary skill in the art ofmedicinal chemistry understands that the total number of suchsubstituents is reasonably limited by the desired properties of thecompound intended. Such properties include, by way of example and notlimitation, physical properties such as molecular weight, solubility orlog P, application properties such as activity against the intendedtarget, and practical properties such as ease of synthesis.

By way of example and not limitation, R⁹, R¹³ and R¹⁰ are recursivesubstituents in certain embodiments. Typically, each of these mayindependently occur 20, 19, 18, 17, 16, 15, 14, 13, 12, 1, 10, 9, 8, 7,6, 5, 4, 3, 2, 1, or 0, times in a given embodiment. More typically,each of these may independently occur 12 or fewer times in a givenembodiment. More typically yet, R⁹ will occur 0 to 8 times in a givenembodiment, R¹³ will occur 0 to 6 times in a given embodiment and R¹⁰will occur 0 to 6 times in a given embodiment. Even more typically yet,R⁹ will occur 0 to 6 times in a given embodiment, R¹³ will occur 0 to 4times in a given embodiment and R¹⁰ will occur 0 to 4 times in a givenembodiment.

Recursive substituents are an intended aspect of the invention. One ofordinary skill in the art of medicinal chemistry understands theversatility of such substituents. To the degree that recursivesubstituents are present in an embodiment of the invention, the totalnumber will be determined as set forth above.

Each -Het- is, independently, —N(R⁷)—, —N(R¹⁰)—, —S—, —SO—, —SO₂—; —O—,—SO₂NH—, —NHSO₂—, —NR⁷CO—, —CONR⁷—, —N(R¹³)—, —SO₂NR¹³—, —NR¹³CO—, or—CONR¹³—. In a preferred embodiment, -Het- is —O—, —N(R⁷)—, or —N(R¹⁰)—.Most preferably, -Het- is —O—.

Each -Link- is, independently, —O—, —(CH₂)_(n)—, —O(CH₂)_(m)—,—NR¹³—C(═O)—NR¹³—, —NR¹³—C(═O)—(CH₂)_(m)—, —C(═O)NR¹³—(CH₂)_(m) ⁻,—(CH₂)_(n)-(Z)_(g)—(CH₂)_(n) ⁻, —S—, —SO—, —SO₂—, —SO₂NR⁷—, SO₂NR¹⁰—, or-Het-. In a preferred embodiment, -Link- is —O—, —(CH₂)_(n)—,—NR¹³—C(═O)—(CH₂)_(m)—, or —C(═O)NR¹³—(CH₂)_(m) ⁻.

Each -CAP is, independently, each CAP is,

In a preferred embodiment, CAP is

Each Ar is, independently, phenyl, substituted phenyl, wherein thesubstituents of the substituted phenyl are 1-3 substituentsindependently selected from the group consisting of OH, OCH₃, NR¹³R¹³,Cl, F, and CH₃, or heteroaryl.

Examples of heteroaryl include pyridinyl, pyrazinyl, furanyl, thienyl,tetrazolyl, thiazolidinedionyl, imidazoyl, pyrrolyl, quinolinyl,indolyl, adeninyl, pyrazolyl, thiazolyl, isoxazolyl, benzimidazolyl,purinyl, isoquinolinyl, pyridazinyl, pyrimidinyl, 1,2,3-triazinyl,1,2,4-triazinyl, 1,3,5-triazinyl, cinnolinyl, phthalazinyl,quinazolinyl, quinoxalinyl, and pterdinyl groups.

Each W is, independently, thiazolidinedione, oxazolidinedione,heteroaryl-C(═O)NR¹³R¹³, —CN, —O—C(═S)NR¹³R¹³, -(Z)_(g)R¹³,—CR¹⁰((Z)_(g)R¹³)((Z)_(g)R¹³), —C(═O)OAr, —C(═O)NR¹³Ar, imidazoline,tetrazole, tetrazole amide, —SO₂NHR¹³, —SO₂NH—C(R¹³R¹³)—(Z)_(g)—R¹³, acyclic sugar or oligosaccharide, a cyclic amino sugar, oligosaccharide,

There is at least one R⁵ on A¹ and A² and the remaining substituents areR⁶. Each R⁶ is, independently, R⁵, —R⁷, —OR¹¹, —N(R⁷)₂, —(CH₂)_(m)—OR⁸,—O—(CH₂)_(m)—OR⁸, —(CH₂)_(n)—NR⁷R¹⁰, —O—(CH₂)_(m)—NR⁷R¹⁰,—(CH₂)_(n)(CHOR⁸)(CHOR₈)_(n)—CH₂OR⁸,—O—(CH₂)_(m)(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸, —(CH₂CH₂O)_(m)—R⁸,—O—(CH₂CH₂O)_(m)—R⁸, —(CH₂CH₂O)_(m)—CH₂CH₂NR⁷R¹⁰,—O—(CH₂CH₂O)_(m)—CH₂CH₂NR⁷R¹⁰, —(CH₂)_(n)—C(═O)NR⁷R¹⁰,—O—(CH₂)_(m)—C(═O)NR⁷R¹⁰, —(CH₂)_(n)—(Z)_(g)—R⁷,—O—(CH₂)_(m)-(Z)_(g)—R⁷, —(CH₂)_(n)—NR¹⁰—CH₂(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸,—O—(CH₂)_(m)—NR¹⁰—CH₂(CHOR⁸)(CHOR₈)_(n)—CH₂OR⁸, —(CH₂)_(n)—CO₂R⁷,—O—(CH₂)_(m)—CO₂R⁷, —OSO₃H, —O-glucuronide, —O-glucose,

When two R⁶ are —OR¹¹ and are located adjacent to each other on thearomatic carbocycle or aromatic heterocycle, the two OR¹¹ may form amethylenedioxy group; i.e., a group of the formula —O—CH₂—O—.

In addition, one or more of the R⁶ groups can be one of the R⁵ groupswhich fall within the broad definition of R⁶ set forth above.

R⁶ may be hydrogen. Therefore, provided that the aromatic carbocycle oraromatic heterocycle is substituted with R⁵, the remaining R⁶ may behydrogen. Preferably, at most, 3 of the R⁶ groups are other thanhydrogen. More preferably, provided that the aromatic carbocycle oraromatic heterocycle is substituted with R⁵, then R⁶ is H.

Each g is, independently, an integer from 1 to 6. Therefore, each g maybe 1, 2, 3, 4, 5, or 6.

Each m is an integer from 1 to 7. Therefore, each m may be 1, 2, 3, 4,5, 6, or 7.

Each n is an integer from 0 to 7. Therefore, each n may be 0, 1, 2, 3,4, 5, 6, or 7.

Each Z is, independently, —(CHOH)—, —C(═O)—, —(CHNR⁷R¹⁰)—, —(C═NR¹⁰)—,—NR¹⁰—, —(CH₂)_(n)—, —(CHNR¹³R¹³)—, —(C═NR¹³)—, or —NR¹³—. As designatedby (Z)_(g) in certain embodiments, Z may occur one, two, three, four,five or six times and each occurrence of Z is, independently, —(CHOH)—,—C(═O)—, —(CHNR⁷R¹⁰)—, —(C═NR¹⁰)—, —NR¹⁰—, —(CH₂)_(n)—, —(CHNR¹³R¹³)—,—(C═NR¹³)—, or —NR¹³—. Therefore, by way of example and not by way oflimitation, (Z)_(g) can be —(CHOH)—(CHNR⁷R¹⁰)—,—(CHOH)—(CHNR⁷R¹⁰)—C(═O)—, —(CHOH)—(CHNR⁷R¹⁰)—C(═O)—(CH₂)_(n)—,—(CHOH)—(CHNR⁷R¹⁰)—C(═O)—(CH₂)_(n)—(CHNR¹³R¹³)—,—(CHOH)—(CHNR⁷R¹⁰)—C(═O)—(CH₂)_(n)—(CHNR¹³R¹³)—C(═O)—, and the like.

In any variable containing —CHOR⁸— or —CH₂OR⁸ groups, when any —CHOR⁸—or —CH₂OR⁸ groups are located 1,2- or 1,3- with respect to each other,the R⁸ groups may, optionally, be taken together to form a cyclic mono-or di-substituted 1,3-dioxane or 1,3-dioxolane.

More specific examples of suitable compounds represented by formula (I)are shown in formula II below wherein A¹ is defined as above:

In a preferred aspect of formula II, A¹ is selected from phenyl,indenyl, napthalenyl, 1,2-dihydronapthalenyl,1,2,3,4-tetrahydronapthalenyl, anthracenyl, fluorenyl, phenanthrenyl,azulenyl, cyclohepta-1,3,5-trienyl or 5H-dibenzo[a,d]cycloheptenyl.

In another preferred aspect of formula II, A¹ is

wherein each Q is, independently, C—H, C—R⁵, or C—R⁶, with the provisothat at least one Q is C—R⁵. Preferably, 4 Q are C—H. Preferably, eachR⁶ is H. Preferably, R⁵ is -Link-(CH₂)_(m)-CAP,-Link-(CH₂)_(n)(CHOR⁸)(CHOR⁸)_(n)-CAP, -Link-(CH₂CH₂O)_(m)—CH₂-CAP,-Link-(CH₂CH₂O)_(m)—CH₂CH₂-CAP, -Link-(CH₂)_(m)-(Z)_(g)-CAP,-Link-(CH₂)_(n)(Z)_(g)—(CH₂)_(m)-CAP,-Link-(CH₂)_(n)—NR¹³—CH₂(CHOR⁸)(CHOR⁸)_(n)-CAP,-Link-(CH₂)_(n)—(CHOR⁸)_(m)CH₂—NR¹³-(Z)_(g)-CAP,-Link-(CH₂)_(n)NR¹³—(CH₂)_(m)(CHOR⁸)_(n)CH₂NR¹³-(Z)_(g)-CAP,-Link-(CH₂)_(m)-(Z)_(g)—(CH₂)_(m)-CAP, -Link-NH—C(═O)—NH—(CH₂)_(m)-CAP,-Link-(CH₂)_(m)—C(═O)NR¹³—(CH₂)_(m)-CAP,-Link-(CH₂)_(n)-(Z)_(g)—(CH₂)_(m)-(Z)_(g)-CAP, or-Link-Z_(g)-(CH₂)_(m)-Het-(CH₂)_(m)-CAP;

Most preferably, R⁵ is

and four Q are C—H.

In another preferred aspect of formula II, A¹ is

Preferably. R⁵ is -Link-(CH₂)_(m)-CAP,-Link-(CH₂)_(n)(CHOR⁸)(CHOR⁸)_(n)-CAP, -Link-(CH₂CH₂O)_(m)—CH₂-CAP,-Link-(CH₂CH₂O)_(m)—CH₂CH₂-CAP, -Link-(CH₂)_(m)-(Z)_(g)-CAP,-Link-(CH₂)_(n)(Z)_(g)—(CH₂)_(m)-CAP,-Link-(CH₂)_(n)—NR¹³—CH₂(CHOR⁸)(CHOR⁸)_(n)-CAP,-Link-(CH₂)_(n)—(CHOR⁸)_(m)CH₂NR¹³-(Z)_(g)-CAP,-Link-(CH₂)_(n)NR¹³—(CH₂)_(m)(CHOR⁸)_(n)CH₂NR¹³—(Z)_(g)-CAP,-Link-(CH₂)_(m)-(Z)_(g)—(CH₂)_(m)-CAP, -Link-NH—C(═O)—NH—CH₂)_(m)-CAP,-Link-(CH₂)_(m)—C(═O)NR¹³—(CH₂)_(m)-CAP,-Link-(CH₂)_(n)-(Z)_(g)—(CH₂)_(m)-(Z)_(g)-CAP, or-Link-Z_(g)-(CH₂)_(m)-Het-(CH₂)_(m)-CAP;

Most preferably, R⁵ is

In a particularly preferred embodiment, the compounds of formula I,formula II, or formula III are:

The compounds described herein may be prepared and used as the freebase. Alternatively, the compounds may be prepared and used as apharmaceutically acceptable salt. Pharmaceutically acceptable salts aresalts that retain or enhance the desired biological activity of theparent compound and do not impart undesired toxicological effects.Examples of such salts are (a) acid addition salts formed with inorganicacids, for example, hydrochloric acid, hydrobromic acid, sulfuric acid,phosphoric acid, nitric acid and the like; (b) salts formed with organicacids such as, for example, acetic acid, oxalic acid, tartaric acid,succinic acid, maleic acid, fumaric acid, gluconic acid, citric acid,malic acid, ascorbic acid, benzoic acid, tannic acid, palmitic acid,alginic acid, polyglutamic acid, naphthalenesulfonic acid,methanesulfonic acid, p-toluenesulfonic acid, naphthalenedisulfonicacid, polygalacturonic acid, malonic acid, sulfosalicylic acid, glycolicacid, 2-hydroxy-3-naphthoate, pamoate, salicylic acid, stearic acid,phthalic acid, mandelic acid, lactic acid and the like; and (c) saltsformed from elemental anions for example, chlorine, bromine, and iodine.

It is to be noted that all enantiomers, diastereomers, and racemicmixtures, tautomers, polymorphs, pseudopolymorphs and pharmaceuticallyacceptable salts of compounds within the scope of formula (I), formulaII, or formula Ill are embraced by the present invention. All mixturesof such enantiomers and diastereomers are within the scope of thepresent invention.

A compound of formula I-III and its pharmaceutically acceptable saltsmay exist as different polymorphs or pseudopolymorphs. As used herein,crystalline polymorphism means the ability of a crystalline compound toexist in different crystal structures. The crystalline polymorphism mayresult from differences in crystal packing (packing polymorphism) ordifferences in packing between different conformers of the same molecule(conformational polymorphism). As used herein, crystallinepseudopolymorphism means the ability of a hydrate or solvate of acompound to exist in different crystal structures. The pseudopolymorphsof the instant invention may exist due to differences in crystal packing(packing pseudopolymorphism) or due to differences in packing betweendifferent conformers of the same molecule (conformationalpseudopolymorphism). The instant invention comprises all polymorphs andpseudopolymorphs of the compounds of formula I-III and theirpharmaceutically acceptable salts.

A compound of formula I-III and its pharmaceutically acceptable saltsmay also exist as an amorphous solid. As used herein, an amorphous solidis a solid in which there is no long-range order of the positions of theatoms in the solid. This definition applies as well when the crystalsize is two nanometers or less. Additives, including solvents, may beused to create the amorphous forms of the instant invention. The instantinvention comprises all amorphous forms of the compounds of formulaI-III and their pharmaceutically acceptable salts.

The compounds of formula I-III may exist in different tautomeric forms.One skilled in the art will recognize that amidines, amides, guanidines,ureas, thioureas, heterocycles and the like can exist in tautomericforms. By way of example and not by way of limitation, compounds offormula I-III can exist in various tautomeric forms as shown below:

All possible tautomeric forms of the amidines, amides, guanidines,ureas, thioureas, heterocycles and the like of all of the embodiments offormula I-III are within the scope of the instant invention.

“Enantiomers” refer to two stereoisomers of a compound which arenon-superimposable mirror images of one another.

Stereochemical definitions and conventions used herein generally followS. P. Parker, Ed., McGraw-Hill Dictionary of Chemical Terms (1984)McGraw-Hill Book Company, New York; and Eliel, E. and Wilen, S.,Stereochemistry of Organic Compounds (1994) John Wiley & Sons, Inc., NewYork. Many organic compounds exist in optically active forms, i.e., theyhave the ability to rotate the plane of plane-polarized light. Indescribing an optically active compound, the prefixes D and L or R and Sare used to denote the absolute configuration of the molecule about itschiral center(s). The prefixes d and l, D and L, or (+) and (−) areemployed to designate the sign of rotation of plane-polarized light bythe compound, with S, (−), or l meaning that the compound islevorotatory while a compound prefixed with R, (+), or d isdextrorotatory. For a given chemical structure, these stereoisomers areidentical except that they are mirror images of one another. A specificstereoisomer may also be referred to as an enantiomer, and a mixture ofsuch isomers is often called an enantiomeric mixture. A 50:50 mixture ofenantiomers is referred to as a racemic mixture or a racemate, which mayoccur where there has been no stereoselection or stereospecificity in achemical reaction or process. The terms “racemic mixture” and “racemate”refer to an equimolar mixture of two enantiomeric species, devoid ofoptical activity.

A single stereoisomer, e.g. an enantiomer, substantially free of itsstereoisomer may be obtained by resolution of the racemic mixture usinga method such as formation of diastereomers using optically activeresolving agents (“Stereochemistry of Carbon Compounds,” (1962) by E. L.Eliel, McGraw Hill; Lochmuller, C. H., (1975) J. Chromatogr., 113:(3)283-302). Racemic mixtures of chiral compounds of the invention can beseparated and isolated by any suitable method, including: (1) formationof ionic, diastereomeric salts with chiral compounds and separation byfractional crystallization or other methods, (2) formation ofdiastereomeric compounds with chiral derivatizing reagents, separationof the diastereomers, and conversion to the pure stereoisomers, and (3)separation of the substantially pure or enriched stereoisomers directlyunder chiral conditions.

“Diastereomer” refers to a stereoisomer with two or more centers ofchirality and whose molecules are not mirror images of one another.Diastereomers have different physical properties, e.g. melting points,boiling points, spectral properties, and reactivities. Mixtures ofdiastereomers may separate under high resolution analytical proceduressuch as electrophoresis and chromatography.

Without being limited to any particular theory, it is believed that thecompounds of formula (I), formula II, or formula III function in vivo assodium channel blockers. By blocking epithelial sodium channels presentin mucosal surfaces the compounds of formula (I), formula II, or formulaIII reduce the absorption of water by the mucosal surfaces. This effectincreases the volume of protective liquids on mucosal surfaces,rebalances the system, and thus treats disease.

The present invention also provides methods of treatment that takeadvantage of the properties of the compounds described herein asdiscussed above. The present invention may be used to hydrate mucosalsurfaces including ocular surfaces or surfaces of the eye, airwaysurfaces, gastrointestinal surfaces, oral surfaces, genito-urethralsurfaces, the inner ear and the middle ear. The active compoundsdisclosed herein may be administered in an effective amount to mucosalsurfaces by any suitable means, including topically, orally, rectally,vaginally, ocularly and dermally, etc. For example, for the treatment ofconstipation, the active compounds may be administered orally orrectally to the gastrointestinal mucosal surface. The active compoundmay be combined with a pharmaceutically acceptable carrier in anysuitable form, such as sterile physiological or dilute saline or topicalsolution, as a droplet, tablet or the like for oral administration, as asuppository for rectal or genito-urethral administration, etc.Excipients may be included in the formulation to enhance the solubilityof the active compounds, as desired. Thus, subjects that may be treatedby the methods of the present invention include, but are not limited to,patients afflicted with chronic dry eye, Sjögren's disease, dry mouth(xerostomia), vaginal dryness, cystic fibrosis, primary ciliarydyskinesia, chronic bronchitis, bronchiectasis chronic obstructiveairway disease, artificially ventilated patients, patients with acutepneumonia, etc.

The present invention may be used to obtain a sputum sample from apatient by administering the active compounds to at least one lung of apatient, and then inducing or collecting a sputum sample from thatpatient. Typically, the invention will be administered to respiratorymucosal surfaces via aerosol (liquid or dry powders) or lavage.

Subjects that may be treated by the method of the present invention alsoinclude patients being administered supplemental oxygen nasally (aregimen that tends to dry the airway surfaces); patients afflicted withan allergic disease or response (e.g., an allergic response to pollen,dust, animal hair or particles, insects or insect particles, etc.) thataffects nasal airway surfaces; patients afflicted with a bacterialinfection e.g., staphylococcus infections such as Staphylococcus aureusinfections, Hemophilus influenza infections, Streptococcus pneumoniaeinfections, Pseudomonas acuriginosa infections, etc.) of the nasalairway surfaces; patients afflicted with an inflammatory disease thataffects nasal airway surfaces; or patients afflicted with sinusitis(wherein the active agent or agents are administered to promote drainageof congested mucous secretions in the sinuses by administering an amounteffective to promote drainage of congested fluid in the sinuses), orcombined, rhinosinusitis. The invention may be administered torhino-sinal surfaces by topical delivery, including aerosols and drops.

The present invention is concerned primarily with the treatment of humansubjects, but may also be employed for the treatment of other mammaliansubjects, such as dogs and cats, for veterinary purposes.

As discussed above, the compounds used to prepare the compositions ofthe present invention may be in the form of a pharmaceuticallyacceptable free base. Because the free base of the compound is generallyless soluble in aqueous solutions than the salt, free base compositionsare employed to provide more sustained release of active agent to thelungs. An active agent present in the lungs in particulate form whichhas not dissolved into solution is not available to induce aphysiological response, but serves as a depot of bioavailable drug whichgradually dissolves into solution.

Another aspect of the present invention is a pharmaceutical composition,comprising a compound of formula (I) in a pharmaceutically acceptablecarrier (e.g., an aqueous carrier solution). In general, the compound offormula (I) is included in the composition in an amount effective toinhibit the reabsorption of water by mucosal surfaces.

Pharmaceutically acceptable carriers for ophthalmic indications includesolutions, emulsions, suspensions, and sustained release formsincluding, but not limited to, dissolvable inserts, plugs, or contactlenses. Pharmaceutically acceptable carriers include, but are notlimited to buffers (including phosphate, citrate, bicarbonate, andborate); tonicity adjusting agents (sodium chloride, potassium chloride,Mannitol, dextrose); viscosity enhancing agents (carboxymethylcellulose, glycerol). Pharmaceutically acceptable carriers can besterile or preserved with agents including, but not limited tobenzalkonium chloride.

Without being limited to any particular theory, it is believed thatsodium channel blockers of the present invention block epithelial sodiumchannels present in mucosal surfaces. The sodium channel blockerdescribed herein reduces the absorption of salt and water by the mucosalsurfaces. This effect increases the volume of protective liquids onmucosal surfaces, rebalances the system, and thus treats disease.

Uses

The compounds of the invention exhibit activity as sodium channelblockers. Without being bound by any particular theory, it is believedthat the compounds of the invention may function in vivo by blockingepithelial sodium channels present in mucosal surfaces and therebyreduce the absorption of water by the mucosal surfaces. This effectincreases the volume of protective liquids on mucosal surfaces, andrebalances the system.

As a consequence, the compounds of the invention are useful asmedicaments, particularly for the treatment of clinical conditions forwhich a sodium channel blocker may be indicated. Sodium channel blockersmay be indicated for the treatment of conditions which are amelioratedby increased mucosal hydration in mucosal surfaces other than pulmonarymucosal surfaces. Examples of such conditions include dry mouth(xerostomia), dry skin, vaginal dryness, sinusitis, rhinosinusitis,nasal dehydration, including nasal dehydration brought on byadministering dry oxygen, dry eye, Sjogren's disease, otitis media,primary ciliary dyskinesia, distal intestinal obstruction syndrome,esophagitis, constipation, and chronic diverticulitis. The compounds ofthe invention can also be used for promoting ocular or cornealhydration.

Other conditions that may benefit from treatment with a sodium channelblocker include pulmonary conditions, such as diseases associated withreversible or irreversible airway obstruction, chronic obstructivepulmonary disease (COPD), including acute exacerbations of COPD, asthma,bronchiectasis (including bronchiectasis due to conditions other thancystic fibrosis), acute bronchitis, chronic bronchitis, post-viralcough, cystic fibrosis, emphysema, pneumonia, panbronchiolitis, andtransplant-associated bronchiolitis, including lung- and bonemarrow-transplant associated bronchiolitis, in a human in need thereof.The compounds of the invention may also be useful for treatingventilator-associated tracheobronchitis and/or preventingventilator-associated pneumonia in ventilated patients. The presentinvention comprises methods for treating each of these conditions in amammal in need thereof, preferably in a human in need thereof, eachmethod comprising administering to said mammal a pharmaceuticallyeffective amount of a compound of the present invention, or apharmaceutically acceptable salt thereof. Also provided are (a) a methodfor reducing exacerbations of COPD in a mammal in need thereof; (b) amethod for reducing exacerbations of CF in a mammal in need thereof; (c)a method of improving lung function (FEV1) in a mammal in need thereof,(d) a method of improving lung function (FEV1) in a mammal experiencingCOPD, (e) a method of improving lung function (FEV1) in a mammalexperiencing CF, (f) a method of reducing airway infections in a mammalin need thereof.

Also provided is a method of stimulating, enhancing or improvingmucociliary clearance in a mammal, the method comprising administeringto a mammal in need thereof a pharmaceutically effective amount of acompound of formula (I), or a pharmaceutically acceptable salt thereof.Mucociliary clearance will be understood to include the naturalmucociliary actions involved in the transfer or clearance of mucus inthe airways, including the self-clearing mechanisms of the bronchi.Therefore, also provided is a method of improving mucus clearance in theairways of a mammal in need thereof.

The compounds of the present invention may also be useful in methods forobtaining a sputum sample from a human. The method may be carried out byadministering a compound of the invention to at least one lung of thepatient, and then inducing and collecting a sputum sample from thathuman.

Accordingly, in one aspect, the present invention provides a method forthe treatment of a condition in a mammal, such as a human, for which asodium channel blocker is indicated.

In other embodiments, the present invention provides each of the methodsdescribed herein with the additional benefit of minimizing oreliminating hyperkalemia in the recipient of the method. Also providedare embodiments comprising each of the methods described herein whereinan improved therapeutic index is achieved.

The terms “treat”, “treating” and “treatment”, as used herein refers toreversing, alleviating, inhibiting the progress of, or preventing thedisorder or condition or one or more symptoms of such disorder orcondition.

All therapeutic methods described herein are carried out byadministering an effective amount of a compound of the invention, acompound of Formula I or a pharmaceutically acceptable salt thereof, toa subject (typically mammal and preferably human) in need of treatment.

In one embodiment the invention provides a method for the treatment of acondition which is ameliorated by increased mucosal hydration in amammal, particularly a human in need thereof. In one embodiment theinvention provides a method for the treatment of a disease associatedwith reversible or irreversible airway obstruction in a mammal,particularly a human, in need thereof. In one particular embodiment thepresent invention provides a method for the treatment of chronicobstructive pulmonary disease (COPD) in a mammal, particularly a humanin need thereof. In one particular embodiment the present inventionprovides a method for reducing the frequency, severity or duration ofacute exacerbation of COPD or for the treatment of one or more symptomsof acute exacerbation of COPD in a mammal, particularly a human in needthereof. In one embodiment the invention provides a method for thetreatment of asthma in a mammal, particularly a human, in need thereof.In one embodiment the invention provides a method for the treatment ofbronchiectasis (including bronchiectasis due to conditions other thancystic fibrosis) in a mammal, particularly a human, in need thereof. Inone embodiment the invention provides a method for the treatment ofbronchitis, including acute and chronic bronchitis in a mammal,particularly a human, in need thereof. In one embodiment the inventionprovides a method for the treatment of post-viral cough in a mammal,particularly a human, in need thereof. In one embodiment the inventionprovides a method for the treatment of cystic fibrosis in a mammal,particularly a human, in need thereof. In one embodiment the inventionprovides a method for the treatment of emphysema in a mammal,particularly a human in need thereof. In one embodiment the inventionprovides a method for the treatment of pneumonia in a mammal,particularly a human in need thereof. In one embodiment the inventionprovides a method for the treatment of panbronchiolitis in a mammal,particularly a human in need thereof. In one embodiment the inventionprovides a method for the treatment of transplant-associatedbronchiolitis, including lung- and bone marrow-transplant associatedbronchiolitis in a mammal, particularly a human in need thereof. In oneembodiment the invention provides a method for treatingventilator-associated tracheobronchitis and/or preventingventilator-associated pneumonia in a ventilated human in need thereof.

In one embodiment the invention provides a method for the treatment ofdry mouth (xerostomia) in a mammal, particularly a human in needthereof. In one embodiment the invention provides a method for thetreatment of dry skin in a mammal, particularly a human in need thereof.In one embodiment the invention provides a method for the treatment ofvaginal dryness in a mammal, particularly a human in need thereof. Inone embodiment the invention provides a method for the treatment ofsinusitis, rhinosinusitis, or nasal dehydration, including nasaldehydration brought on by administering dry oxygen, in a mammal,particularly a human in need thereof. In one embodiment the inventionprovides a method for the treatment of dry eye, or Sjogren's disease, orpromoting ocular or corneal hydration in a mammal, particularly a humanin need thereof. In one embodiment the invention provides a method forthe treatment of otitis media in a mammal, particularly a human in needthereof. In one embodiment the invention provides a method for thetreatment of primary ciliary dyskinesia, in a mammal, particularly ahuman in need thereof. In one embodiment the invention provides a methodfor the treatment of distal intestinal obstruction syndrome,esophagitis, constipation, or chronic diverticulitis in a mammal,particularly a human in need thereof.

There is also provided a compound of the invention for use in medicaltherapy, particularly for use in the treatment of condition in a mammal,such as a human, for which a sodium channel blocker is indicated. Alltherapeutic uses described herein are carried out by administering aneffective amount of a compound of the invention to the subject in needof treatment. In one embodiment there is provided a compound of theinvention for use in the treatment of a pulmonary condition such as adisease associated with reversible or irreversible airway obstruction ina mammal, particularly a human, in need thereof. In one particularembodiment there is provided a compound of the invention for use in thetreatment of chronic obstructive pulmonary disease (COPD) in a mammal,particularly a human in need thereof. In one embodiment, there isprovided a compound of the invention for use in reducing the frequency,severity or duration of acute exacerbation of COPD or for the treatmentof one or more symptoms of acute exacerbation of COPD, in a mammal,particularly a human, in need thereof. In one embodiment there isprovided a compound of the invention for use in the treatment of asthmain a mammal, particularly a human, in need thereof. In one embodimentthere is provided a compound for use in the treatment of bronchiectasis,including bronchiectasis due to conditions other than cystic fibrosis,or bronchitis, including acute bronchitis and chronic bronchitis, in amammal, particularly a human, in need thereof. In one embodiment thereis provided a compound for use in the treatment of post-viral cough, ina mammal, particularly a human, in need thereof. In one embodiment thereis provided a compound for use in the treatment of cystic fibrosis in amammal, particularly a human in need thereof. In one embodiment there isprovided a compound of the invention for use in the treatment ofemphysema in a mammal, particularly a human, in need thereof. In oneembodiment there is provided a compound of the invention for use in thetreatment of pneumonia in a mammal, particularly a human, in needthereof. In one embodiment there is provided a compound of the inventionfor use in the treatment of panbronchiolitis or transplant-associatedbronchiolitis, including lung- and bone marrow-transplant associatedbronchiolitis in a mammal, particularly a human, in need thereof. In oneembodiment there is provided a compound of the invention for use in thetreatment of ventilator-associated tracheobronchitis or preventingventilator-associated pneumonia in a ventilated human in need thereof.

In one embodiment there is provided a compound of the invention for usein the treatment of a condition ameliorated by increased mucosalhydration in mucosal surfaces of a mammal, particularly a human, in needthereof. In one embodiment there is provided a compound for use in thetreatment of dry mouth (xerostomia) in a mammal, particularly a human,in need thereof. In one embodiment there is provided a compound for usein the treatment of dry skin in a mammal, particularly a human, in needthereof. In one embodiment there is provided a compound for use in thetreatment of vaginal dryness in a mammal, particularly a human in needthereof. In one embodiment there is provided a compound of the inventionfor use in the treatment of sinusitis, rhinosinusitis, or nasaldehydration, including nasal dehydration brought on by administering dryoxygen in a mammal, particularly a human, in need thereof. In oneembodiment there is provided a compound of the invention for use in thetreatment of dry eye, or Sjogren's disease or promoting ocular orcorneal hydration in a mammal, particularly a human, in need thereof. Inone embodiment there is provided a compound of the invention for use inthe treatment of otitis media in a mammal, particularly a human, in needthereof. In one embodiment there is provided a compound of the inventionfor use in the treatment of primary ciliary dyskinesia in a mammal,particularly a human, in need thereof. In one embodiment there isprovided a compound of the invention for use in the treatment of distalintestinal obstruction syndrome, esophagitis, constipation, or chronicdiverticulitis in a mammal, particularly a human, in need thereof.

The present invention also provides the use of a compound of theinvention in the manufacture of a medicament for the treatment of acondition in a mammal, such as a human, for which a sodium channelblocker is indicated. In one embodiment is provided the use of acompound of the invention in the manufacture of a medicament for thetreatment of diseases associated with reversible or irreversible airwayobstruction, chronic obstructive pulmonary disease (COPD), acuteexacerbations of COPD, asthma, bronchiectasis (including bronchiectasisdue to conditions other than cystic fibrosis), bronchitis (includingacute bronchitis and chronic bronchitis), post-viral cough, cysticfibrosis, emphysema, pneumonia, panbronchiolitis, transplant-associatedbronchiolitis, (including lung- and bone marrow-transplant associatedbronchiolitis), ventilator-associated tracheobronchitis or preventingventilator-associated pneumonia.

In one particular embodiment is provided the use of a compound of theinvention in the manufacture of a medicament for the treatment of acondition ameliorated by increased mucosal hydration in mucosalsurfaces, treatment of dry mouth (xerostomia), dry skin, vaginaldryness, sinusitis, rhinosinusitis, nasal dehydration, including nasaldehydration brought on by administering dry oxygen, treatment of dryeye, Sjogren's disease, promoting ocular or corneal hydration, treatmentof otitis media, primary ciliary dyskinesia, distal intestinalobstruction syndrome, esophagitis, constipation, or chronicdiverticulitis

The terms “effective amount”, “pharmaceutically effective amount”,“effective dose”, and “pharmaceutically effective dose” as used herein,refer to an amount of compound of the invention which is sufficient inthe subject to which it is administered, to elicit the biological ormedical response of a cell culture, tissue, system, or mammal (includinghuman) that is being sought, for instance by a researcher or clinician.The term also includes within its scope, amounts effective to enhancenormal physiological function. In one embodiment, the effective amountis the amount needed to provide a desired level of drug in thesecretions and tissues of the airways and lungs, or alternatively, inthe bloodstream of a subject to be treated to give an anticipatedphysiological response or desired biological effect when such acomposition is administered by inhalation. For example an effectiveamount of a compound of the invention for the treatment of a conditionfor which a sodium channel blocker is indicated is sufficient in thesubject to which it is administered to treat the particular condition.In one embodiment an effective amount is an amount of a compound of theinvention which is sufficient for the treatment of COPD or cysticfibrosis in a human.

The precise effective amount of the compounds of the invention willdepend on a number of factors including but not limited to the species,age and weight of the subject being treated, the precise conditionrequiring treatment and its severity, the bioavailability, potency, andother properties of the specific compound being administered, the natureof the formulation, the route of administration, and the deliverydevice, and will ultimately be at the discretion of the attendantphysician or veterinarian. Further guidance with respect to appropriatedose may be found in considering conventional dosing of other sodiumchannel blockers, such as amiloride, with due consideration also beinggiven to any differences in potency between amiloride and the compoundsof the present invention.

A pharmaceutically effective dose administered topically to the ocularsurfaces of a subject (e.g., by applied as a topical eye drop) of acompound of the invention for treatment of a 70 kg human may be in therange of from about 0.01 to about 1000 μg.

A pharmaceutically effective dose administered topically to the airwaysurfaces of a subject (e.g., by inhalation) of a compound of theinvention for treatment of a 70 kg human may be in the range of fromabout 0.1 to about 1000 μg. Typically, the daily dose administeredtopically to the airway surfaces will be an amount sufficient to achievedissolved concentration of active agent on the airway surfaces of fromabout 10⁻⁹, 10⁻⁸, or 10⁻⁷ to about 10⁻⁴, 10⁻³, 10⁻², or 10⁻¹Moles/liter, more preferably from about 10⁻⁹ to about 10⁻⁴ Moles/liter.The selection of the specific dose for a patient will be determined bythe attendant physician, clinician or veterinarian of ordinary skill inthe art based upon a number of factors including those noted above. Inone particular embodiment the dose of a compound of the invention forthe treatment of a 70 kg human will be in the range of from about 0.1 toabout 1,000 μg. In one embodiment, the dose of a compound of theinvention for the treatment of a 70 kg human will be in the range offrom about 0.5 to about 50 μg. In another embodiment, thepharmaceutically effective dose will be from about 1 to about 10 μg. Inanother embodiment, the pharmaceutically effective dose will be fromabout 10 μg to about 40 μg. In a further embodiment, thepharmaceutically effective dose will be from about 15 μg to about 30 μg.The foregoing suggested doses may be adjusted using conventional dosecalculations if the compound is administered via a different route.Determination of an appropriate dose for administration by other routesis within the skill of those in the art in light of the foregoingdescription and the general knowledge in the art. These doses andsolutions will range from about 0.00001% to 10% on a weight per volume(w/v) basis.

Delivery of an effective amount of a compound of the invention mayentail delivery of a single dosage form or multiple unit doses which maybe delivered contemporaneously or separate in time over a designatedperiod, such as 24 hours. A dose of a compound of the invention (aloneor in the form of a composition comprising the same) may be administeredfrom one to ten times per day. Typically, a compound of the invention(alone or in the form of a composition comprising the same) will beadministered four, three, two, or once per day (24 hours).

The compounds of formula (I) of the present invention are also usefulfor treating airborne infections. Examples of airborne infectionsinclude, for example, RSV. The compounds of formula (I) of the presentinvention are also useful for treating an anthrax infection. The presentinvention relates to the use of the compounds of formula (I) of thepresent invention for prophylactic, post-exposure prophylactic,preventive or therapeutic treatment against diseases or conditionscaused by pathogens. In a preferred embodiment, the present inventionrelates to the use of the compounds of formula (I) for prophylactic,post-exposure prophylactic, preventive or therapeutic treatment againstdiseases or conditions caused by pathogens which may be used inbioterrorism.

In recent years, a variety of research programs and biodefense measureshave been put into place to deal with concerns about the use ofbiological agents in acts of terrorism. These measures are intended toaddress concerns regarding bioterrorism or the use of microorganisms orbiological toxins to kill people, spread fear, and disrupt society. Forexample, the National Institute of Allergy and Infectious Diseases(NIAID) has developed a Strategic Plan for Biodefense Research whichoutlines plans for addressing research needs in the broad area ofbioterrorism and emerging and reemerging infectious diseases. Accordingto the plan, the deliberate exposure of the civilian population of theUnited States to Bacillus anthracis spores revealed a gap in thenation's overall preparedness against bioterrorism. Moreover, the reportdetails that these attacks uncovered an unmet need for tests to rapidlydiagnose, vaccines and immunotherapies to prevent, and drugs andbiologics to cure disease caused by agents of bioterrorism.

Much of the focus of the various research efforts has been directed tostudying the biology of the pathogens identified as potentiallydangerous as bioterrorism agents, studying the host response againstsuch agents, developing vaccines against infectious diseases, evaluatingthe therapeutics currently available and under investigation againstsuch agents, and developing diagnostics to identify signs and symptomsof threatening agents. Such efforts are laudable but, given the largenumber of pathogens which have been identified as potentially availablefor bioterrorism, these efforts have not yet been able to providesatisfactory responses for all possible bioterrorism threats.Additionally, many of the pathogens identified as potentially dangerousas agents of bioterrorism do not provide adequate economic incentivesfor the development of therapeutic or preventive measures by industry.Moreover, even if preventive measures such as vaccines were availablefor each pathogen which may be used in bioterrorism, the cost ofadministering all such vaccines to the general population isprohibitive.

Until convenient and effective treatments are available against everybioterrorism threat, there exists a strong need for preventative,prophylactic or therapeutic treatments which can prevent or reduce therisk of infection from pathogenic agents.

The present invention provides such methods of prophylactic treatment.In one aspect, a prophylactic treatment method is provided comprisingadministering a prophylactically effective amount of the compounds offormula (I) to an individual in need of prophylactic treatment againstinfection from one or more airborne pathogens. A particular example ofan airborne pathogen is anthrax.

In another aspect, a prophylactic treatment method is provided forreducing the risk of infection from an airborne pathogen which can causea disease in a human, said method comprising administering an effectiveamount of the compounds of formula (I) to the lungs of the human who maybe at risk of infection from the airborne pathogen but is asymptomaticfor the disease, wherein the effective amount of a sodium channelblocker and osmolye are sufficient to reduce the risk of infection inthe human. A particular example of an airborne pathogen is anthrax.

In another aspect, a post-exposure prophylactic treatment or therapeutictreatment method is provided for treating infection from an airbornepathogen comprising administering an effective amount of the compoundsof formula (I) to the lungs of an individual in need of such treatmentagainst infection from an airborne pathogen. The pathogens which may beprotected against by the prophylactic post exposure, rescue andtherapeutic treatment methods of the invention include any pathogenswhich may enter the body through the mouth, nose or nasal airways, thusproceeding into the lungs. Typically, the pathogens will be airbornepathogens, either naturally occurring or by aerosolization. Thepathogens may be naturally occurring or may have been introduced intothe environment intentionally after aerosolization or other method ofintroducing the pathogens into the environment. Many pathogens which arenot naturally transmitted in the air have been or may be aerosolized foruse in bioterrorism. The pathogens for which the treatment of theinvention may be useful includes, but is not limited to, category A, Band C priority pathogens as set forth by the NIAID. These categoriescorrespond generally to the lists compiled by the Centers for DiseaseControl and Prevention (CDC). As set up by the CDC, Category A agentsare those that can be easily disseminated or transmittedperson-to-person, cause high mortality, with potential for major publichealth impact. Category B agents are next in priority and include thosethat are moderately easy to disseminate and cause moderate morbidity andlow mortality. Category C consists of emerging pathogens that could beengineered for mass dissemination in the future because of theiravailability, ease of production and dissemination and potential forhigh morbidity and mortality. Particular examples of these pathogens areanthrax and plague. Additional pathogens which may be protected againstor the infection risk therefrom reduced include influenza viruses,rhinoviruses, adenoviruses and respiratory syncytial viruses, and thelike. A further pathogen which may be protected against is thecoronavirus which is believed to cause severe acute respiratory syndrome(SARS).

The present invention also relates to the use of sodium channel blockersof Formula I, or a pharmaceutically acceptable salt thereof, forpreventing, mitigating, and/or treating deterministic health effects tothe respiratory tract caused by exposure to radiological materials,particularly respirable aerosols containing radionuclides from nuclearattacks, such as detonation of radiological dispersal devices (RDD), oraccidents, such as nuclear power plant disasters. As such, providedherein is a method for preventing, mitigating, and/or treatingdeterministic health effects to the respiratory tract and/or otherbodily organs caused by respirable aerosols containing radionuclides ina recipient in need thereof, including in a human in need thereof, saidmethod comprising administering to said human an effective amount of acompound of Formula (I), or a pharmaceutically acceptable salt thereof.

A major concern associated with consequence management planning forexposures of members of the public to respirable aerosols containingradionuclides from nuclear attacks, such as detonation of radiologicaldispersal devices (RDD), or accidents, such as nuclear power plantdisasters is how to prevent, mitigate or treat potential deterministichealth effects to the respiratory tract, primarily the lung. It isnecessary to have drugs, techniques and procedures, and trainedpersonnel prepared to manage and treat such highly internallycontaminated individuals.

Research has been conducted to determine ways in which to prevent,mitigate or treat potential damage to the respiratory tract and variousorgans in the body that is caused by internally deposited radionuclides.To date, most of the research attention has focused on strategiesdesigned to mitigate health effects from internally depositedradionuclides by accelerating their excretion or removal. Thesestrategies have focused on soluble chemical forms that are capable ofreaching the blood stream and are deposited at remote systemic sitesspecific to a given radioelement. Such approaches will not work in caseswhere the deposited radionuclide is in relatively insoluble form.Studies have shown that many, if not most of the physicochemical formsof dispersed radionuclides from RDDs, will be in relatively insolubleform.

The only method known to effectively reduce the radiation dose to thelungs from inhaled insoluble radioactive aerosols is bronchoalveolarlavage or BAL. This technique, which was adapted from that already inuse for the treatment of patients with alveolar proteinosis, has beenshown to be a safe, repeatable procedure, even when performed over anextended period of time. Although there are variations in procedure, thebasic method for BAL is to anaesthetize the subject, followed by theslow introduction of isotonic saline into a single lobe of the lunguntil the function residual capacity is reached. Additional volumes arethen added and drained by gravity.

The results of studies using BAL on animals indicate that about 40% ofthe deep lung content can be removed by a reasonable sequence of BALs.In some studies, there was considerable variability among animals in theamount of radionuclide recovered. The reasons for the variability arecurrently not understood.

Further, based on a study on animals, it is believed that a significantdose reduction from BAL therapy results in mitigation of health effectsdue to inhalation of insoluble radionuclides. In the study, adult dogsinhaled insoluble ¹⁴⁴Ce-FAP particles. Two groups of dogs were givenlung contents of ¹⁴⁴Ce known to cause radiation pneumonitis andpulmonary fibrosis (about 2 MBq/kg body mass), with one group beingtreated with 10 unilateral lavages between 2 and 56 days after exposure,the other untreated. A third group was exposed at a level of ¹⁴⁴Cecomparable to that seen in the BAL-treated group after treatment (about1 MBq/kg), but these animals were untreated. All animals were allowed tolive their lifespans, which extended to 16 years. Because there isvariability in initial lung content of ¹⁴⁴Ce among the dogs in eachgroup, the dose rates and cumulative doses for each group overlap.Nevertheless, the effect of BAL in reducing the risk frompneumonitis/fibrosis was evident from the survival curves. In theuntreated dogs with lung contents of 1.5-2.5 MBq/kg, the mean survivaltime was 370±65 d. For the treated dogs, the mean survival was 1270±240d, which was statistically significantly different. The third group,which received lung contents of ¹⁴⁴Ce of 0.6-1.4 MBq had a mean survivaltime of 1800±230, which was not statistically different from the treatedgroup. Equally important to the increased survival, the dogs in thehigh-dose untreated group died from deterministic effects to lung(pneumonitis/fibrosis) while the treated dogs did not. Instead, thetreated dogs, like the dogs in the low-dose untreated group, mostly hadlung tumors (hemangiosarcoma or carcinoma). Therefore, the reduction indose resulting from BAL treatment appears to have produced biologicaleffects in lung that were predictable based on the radiation doses thatthe lungs received.

Based on these results, it is believed that decreasing the residualradiological dose further by any method or combination of methods forenhancing the clearance of particles from the lung would furtherdecrease the probability of health effects to lung. However, BAL is aprocedure that has many drawbacks. BAL is a highly invasive procedurethat must be performed at specialized medical centers by trainedpulmonologists. As such, a BAL procedure is expensive. Given thedrawbacks of BAL, it is not a treatment option that would be readily andimmediately available to persons in need of accelerated removal ofradioactive particles, for example, in the event of a nuclear attack. Inthe event of a nuclear attack or a nuclear accident, immediate andrelatively easily administered treatment for persons who have beenexposed or who are at risk of being exposed is needed. Sodium channelblockers administered as an inhalation aerosol have been shown torestore hydration of airway surfaces. Such hydration of airway surfacesaids in clearing accumulated mucus secretions and associated particulatematter from the lung. As such, without being bound by any particulartheory, it is believed that sodium channel blockers can be used toaccelerate the removal of radioactive particles from airway passages.

As discussed above, the greatest risk to the lungs following aradiological attack, such as a dirty bomb, results from the inhalationand retention of insoluble radioactive particles. As a result ofradioactive particle retention, the cumulative exposure to the lung issignificantly increased, ultimately resulting in pulmonaryfibrosis/pneumonitis and potentially death. Insoluble particles cannotbe systemically cleared by chelating agents because these particles arenot in solution. To date, the physical removal of particulate matterthrough BAL is the only therapeutic regimen shown to be effective atmitigating radiation-induced lung disease. As discussed above, BAL isnot a realistic treatment solution for reducing the effects ofradioactive particles that have been inhaled into the body. As such, itis desirable to provide a therapeutic regimen that effectively aids inclearing radioactive particles from airway passages and that, unlikeBAL, is relatively simple to administer and scalable in a large-scaleradiation exposure scenario. In addition, it is also desirable that thetherapeutic regimen be readily available to a number of people in arelatively short period of time.

In an aspect of the present invention, a method for preventing,mitigating, and/or treating deterministic health effects to therespiratory tract and/or other bodily organs caused by respirableaerosols containing radionuclides comprises administering an effectiveamount of a sodium channel blocker of Formula I or a pharmaceuticallyacceptable salt thereof to an individual in need. In a feature of thisaspect, the sodium channel blocker is administered in conjunction withan osmolyte. With further regard to this feature, the osmolyte ishypertonic saline (HS). In a further feature, the sodium channel blockerand the osmolyte are administered in conjunction with an ion transportmodulator. With further regard to this feature, the ion transportmodulator may be selected from the group consisting of β-agonists, CFTRpotentiators, purinergic receptor agonists, lubiprostones, and proteaseinhibitors. In another feature of this aspect, the radionuclides areselected from the group consisting of Colbalt-60, Cesium-137,Iridium-192, Radium-226, Phospohrus-32, Strontium-89 and 90, Iodine-125,Thallium-201, Lead-210, Thorium-234, Uranium-238, Plutonium, Cobalt-58,Chromium-51, Americium, and Curium. In a further feature, theradionuclides are from a radioactive disposal device. In yet anotherfeature, the sodium channel blocker or pharmaceutically acceptable saltthereof is administered in an aerosol suspension of respirable particleswhich the individual inhales. In an additional feature, the sodiumchannel blocker or a pharmaceutically acceptable salt thereof isadministered post-exposure to the radionuclides.

Compositons

While it is possible for a compound of the invention to be administeredalone, in some embodiments it is preferable to present it in the form ofa composition, particularly a pharmaceutical composition (formulation).Thus, in another aspect, the invention provides compositions, andparticularly pharmaceutical compositions (such as an inhalablepharmaceutical composition) comprising a pharmaceutically effectiveamount of a compound of the invention as an active ingredient, and apharmaceutically acceptable excipient, diluent or carrier. The term“active ingredient” as employed herein refers to any compound of theinvention or combination of two or more compounds of the invention in apharmaceutical composition. Also provided are specific embodiments inwhich a pharmaceutical composition comprises a pharmaceuticallyeffective amount of a compound of Formula (I), independently or incombination, and a pharmaceutically acceptable excipient, diluent orcarrier.

Also provided is a kit comprising i) a pharmaceutically effective amountof a compound of Formula (I), or a pharmaceutically acceptable saltthereof; ii) one or more pharmaceutically acceptable excipients,carriers, or diluents; iii) instructions for administering the compoundof group i) and the excipients, carriers, or diluents of group ii) to asubject in need thereof; and; iv) a container. A subject in need thereofincludes any subject in need of the methods of treatment describedherein.

In one embodiment a kit comprises i) from about 10 μg to about 40 μg ofa compound of Formula (I), or a pharmaceutically acceptable saltthereof, per dose; ii) from about 1 to about 5 mL of diluent per dose;iii) instructions for administering the compound of group i) and thediluent of group ii) to a subject in need thereof; and; iv) a container.In a further embodiment, the diluent is from about 1 to about 5 mL of asaline solution, as described herein, per dose.

Also provided is a kit comprising i) a solution comprising apharmaceutically effective amount of a compound of Formula (I), or apharmaceutically acceptable salt thereof; dissolved in apharmaceutically acceptable diluent; iii) instructions for administeringthe solution of group i) to a subject in need thereof; and iii) acontainer.

Also provided is a kit comprising i) a solution comprising from about 10μg to about 40 μg of a compound of Formula (I), or a pharmaceuticallyacceptable salt thereof; dissolved in a pharmaceutically acceptablediluent; iii) instructions for administering the solution of group i) toa subject in need thereof; and iii) a container. In a furtherembodiment, the diluent is from about 1 to about 5 mL of a salinesolution, as described herein, per dose.

For each of the kits described above there is an additional embodimentin which the diluent is hypertonic saline.

The pharmaceutically acceptable excipient(s), diluent(s) or carrier(s)must be acceptable in the sense of being compatible with the otheringredients of the formulation and not deleterious to the recipientthereof. Generally, the pharmaceutically acceptable excipient(s),diluent(s) or carrier(s) employed in the pharmaceutical formulation are“non-toxic” meaning that it/they is/are deemed safe for consumption inthe amount delivered in the formulation and “inert” meaning that it/theydoes/do not appreciable react with or result in an undesired effect onthe therapeutic activity of the active ingredient(s). Pharmaceuticallyacceptable excipients, diluents and carriers are conventional in the artand may be selected using conventional techniques, based upon thedesired route of administration. See, REMINGTON'S, PHARMACEUTICALSCIENCES, Lippincott Williams & Wilkins; 21^(st) Ed (May 1, 2005).Preferably, the pharmaceutically acceptable excipient(s), diluent(s) orcarrier(s) are Generally Regarded As Safe (GRAS) according to the FDA.

Pharmaceutical compositions according to the invention include thosesuitable for oral administration, parenteral administration, includingsubcutaneous, intradermal, intramuscular, intravenous andintraarticular; topical administration, including topical administrationto the skin, eyes, ears, etc; vaginal or rectal administration; andadministration to the respiratory tract, including the nasal cavitiesand sinuses, oral and extrathoracic airways, and the lungs, including byuse of aerosols which may be delivered by means of various types of drypowder inhalers, pressurized metered dose inhalers, softmist inhalers,nebulizers, or insufflators. The most suitable route of administrationmay depend upon, several factors including the patient and the conditionor disorder being treated.

The formulations may be presented in unit dosage form or in bulk form asfor example in the case of formulations to be metered by an inhaler andmay be prepared by any of the methods well known in the art of pharmacy.Generally, the methods include the step of bringing the activeingredient into association with the carrier, diluent or excipient andoptionally one or more accessory ingredients. In general theformulations are prepared by uniformly and intimately bringing intoassociation the active ingredient with one or more liquid carriers,diluents or excipients or finely divided solid carriers, diluents orexcipients, or both, and then, if necessary, shaping the product intothe desired formulation.

Pharmaceutical compositions for topical administration may be formulatedas ointments, creams, suspensions, lotions, powders, solutions, pastes,gels, sprays, aerosols or oils. Compositions designed for the treatmentof the eyes or other external tissues, for example the mouth and skin,may be applied as a topical ointment, cream or eye drops. Whenformulated as an ointment, the active ingredient may be employed witheither a paraffinic or a water-miscible ointment base. Alternatively,the active ingredient may be formulated in a cream with an oil-in-watercream base or a water-in-oil base.

Other compositions designed for topical administration to the eyes orears include eye drops and ear drops wherein the active ingredient isdissolved or suspended in a suitable carrier, such as for example anaqueous solvent, including saline.

Formulations suitable for oral administration may be presented asdiscrete units such as capsules, cachets or tablets, each containing apredetermined amount of the active ingredient; as a powder or granules;as a solution or suspension in an aqueous liquid or a non-aqueousliquid; or as an oil-in-water liquid emulsion or a water-in-oil liquidemulsion. The active ingredient may also be presented as a sachet,bolus, electuary or paste.

A tablet may be made by compression or molding, optionally with one ormore accessory ingredients. Compressed tablets may be prepared bycompressing in a suitable machine the active ingredient in afree-flowing form such as a powder or granules, optionally mixed with abinders, lubricant, inert diluent, surface active or dispersing agent.Molded tablets may be made by molding in a suitable machine a mixture ofthe powdered compound moistened with an inert liquid diluent. Thetablets may optionally be coated or scored and may be formulated so asto provide slow or controlled release of the active ingredient therein.

Formulations for topical administration in the mouth, for examplebuccally or sublingually, include lozenges, comprising the activeingredient in a flavored base such as sucrose and acacia or tragacanth,and pastilles comprising the active ingredient in a base such as gelatinand glycerin or sucrose and acacia.

Formulations for parenteral administration include aqueous andnon-aqueous sterile injection solutions which may contain anti-oxidants,buffers, bacteriostats and solutes which render the formulation isotonicwith the blood of the intended recipient; and aqueous and non-aqueoussterile suspensions which may include suspending agents and thickeningagents. The formulations may be presented in unit-dose or multi-dosecontainers, for example sealed ampoules and vials, and may be stored ina freeze-dried (lyophilized) condition requiring only the addition ofthe sterile liquid carrier, for example saline or water-for-injection,immediately prior to use. Extemporaneous injection solutions andsuspensions may be prepared from sterile powders, granules and tabletsof the kind previously described.

Oral fluids such as solutions, syrups and elixirs can be prepared indosage unit form so that a given quantity contains a predeterminedamount of the active ingredient. Syrups can be prepared by dissolvingthe active ingredient in a suitably flavored aqueous solution, whileelixirs are prepared through the use of a pharmaceutically acceptablealcoholic vehicle. Suspensions can be formulated by dispersing theactive ingredient in a pharmaceutically acceptable vehicle. Solubilizersand emulsifiers such as ethoxylated isostearyl alcohols and polyoxyethylene sorbitol ethers, preservatives, flavor additive such aspeppermint oil or natural sweeteners or saccharin or other artificialsweeteners, and the like can also be incorporated into oral liquidcompositions.

Liposome delivery systems such as small unilamellar vesicles, largeunilamellar vesicles and multilamellar vesicles may also be employed asdelivery means for the compounds of the invention. Liposomes may beformed from a variety of phospholipids such as cholesterol, stearylamineand phosphatidylcholines.

Compositions designed for nasal administration include aerosols,solutions, suspensions, sprays, mists and drops. Aerosolableformulations for nasal administration may be formulated in much the sameways as aerosolable formulations for inhalation with the condition thatparticles of non-respirable size will be preferred in formulations fornasal administration. Typically, particles of about 5 microns in size,up to the size of visible droplets may be employed. Thus, for nasaladministration, a particle size in the range of 10-500 m may be used toensure retention in the nasal cavity.

Transdermal patches may also be employed, which are designed to remainin contact with the epidermis of the patient for an extended period oftime and promote the absorption of the active ingredient there through.

Compositions for vaginal or rectal administration include ointments,creams, suppositories and enemas, all of which may be formulated usingconventional techniques.

In one preferred embodiment, the composition is an inhalablepharmaceutical composition which is suitable for inhalation and deliveryto the endobronchial space. Typically, such composition is in the formof an aerosol comprising particles for delivery using a nebulizer,pressurized metered dose inhaler (MDI), softmist inhaler, or dry powderinhaler (DPI). The aerosol formulation used in the methods of thepresent invention may be a liquid (e.g., solution) suitable foradministration by a nebulizer, softmist inhaler, or MDI, or a dry powdersuitable for administration by an MDI or DPI.

Aerosols used to administer medicaments to the respiratory tract aretypically polydisperse; that is they are comprised of particles of manydifferent sizes. The particle size distribution is typically describedby the Mass Median Aerodynamic Diameter (MMAD) and the GeometricStandard Deviation (GSD). For optimum drug delivery to the endobronchialspace the MMAD is in the range from about 1 to about 10 μm andpreferably from about 1 to about 5 μm, and the GSD is less than 3, andpreferably less than about 2. Aerosols having a MMAD above 10 μm aregenerally too large when inhaled to reach the lungs. Aerosols with a GSDgreater than about 3 are not preferred for lung delivery as they delivera high percentage of the medicament to the oral cavity. To achieve theseparticle sizes in powder formulation, the particles of the activeingredient may be size reduced using conventional techniques such asmicronisation or spray drying. Non-limiting examples of other processesor techniques that can be used to produce respirable particles includespray drying, precipitation, supercritical fluid, and freeze drying. Thedesired fraction may be separated out by air classification or sieving.In one embodiment, the particles will be crystalline. For liquidformulations, the particle size is determined by the selection of aparticular model of nebulizer, softmist inhaler, or MDI.

Aerosol particle size distributions are determined using devices wellknown in the art. For example a multi-stage Anderson cascade impactor orother suitable method such as those specifically cited within the USPharmacopoeia Chapter 601 as characterizing devices for aerosols emittedfrom metered-dose and dry powder inhalers.

Dry powder compositions for topical delivery to the lung by inhalationmay be formulated without excipient or carrier and instead includingonly the active ingredients in a dry powder form having a suitableparticle size for inhalation. Dry powder compositions may also contain amix of the active ingredient and a suitable powder base(carrier/diluent/excipient substance) such as mono-, di- orpoly-saccharides (e.g., lactose or starch). Lactose is typically thepreferred excipient for dry powder formulations. When a solid excipientsuch as lactose is employed, generally the particle size of theexcipient will be much greater than the active ingredient to aid thedispersion of the formulation in the inhaler.

Non-limiting examples of dry powder inhalers include reservoirmulti-dose inhalers, pre-metered multi-dose inhalers, capsule-basedinhalers and single-dose disposable inhalers. A reservoir inhalercontains a large number of doses (e.g. 60) in one container. Prior toinhalation, the patient actuates the inhaler which causes the inhaler tometer one dose of medicament from the reservoir and prepare it forinhalation. Examples of reservoir DPIs include but are not limited tothe Turbohaler® by AstraZeneca and the ClickHaler® by Vectura.

In a pre-metered multi-dose inhaler, each individual dose has beenmanufactured in a separate container, and actuation of the inhaler priorto inhalation causes a new dose of drug to be released from itscontainer and prepared for inhalation. Examples of multidose DPIinhalers include but are not limited to Diskus® by GSK, Gyrohaler® byVectura, and Prohaler® by Valois. During inhalation, the inspiratoryflow of the patient accelerates the powder out of the device and intothe oral cavity. For a capsule inhaler, the formulation is in a capsuleand stored outside the inhaler. The patient puts a capsule in theinhaler, actuates the inhaler (punctures the capsule), then inhales.Examples include the Rotohaler™ (GlaxoSmithKline), Spinhaler™(Novartis), HandiHaler™ (IB), TurboSpin™ (PH&T). With single-dosedisposable inhalers, the patient actuates the inhaler to prepare it forinhalation, inhales, then disposes of the inhaler and packaging.Examples include the Twincer™ (U Groningen), OneDose™ (GFE), and MantaInhaler™ (Manta Devices).

Generally, dry powder inhalers utilize turbulent flow characteristics ofthe powder path to cause the excipient-drug aggregates to disperse, andthe particles of active ingredient are deposited in the lungs. However,certain dry powder inhalers utilize a cyclone dispersion chamber toproduce particles of the desired respirable size. In a cyclonedispersion chamber, the drug enters a coin shaped dispersion chambertangentially so that the air path and drug move along the outer circularwall. As the drug formulation moves along this circular wall it bouncesaround and agglomerates are broken apart by impact forces. The air pathspirals towards the center of the chamber exiting vertically. Particlesthat have small enough aerodynamic sizes can follow the air path andexit the chamber. In effect, the dispersion chamber works like a smalljet mill. Depending on the specifics of the formulation, large lactoseparticles may be added to the formulation to aid in the dispersionthrough impact with the API particles.

The Twincer™ single-dose disposable inhaler appears to operate using acoin-shaped cyclone dispersion chamber referred to as an “airclassifier.” See, U.S. Published Patent Application No. 2006/0237010 toRijksuniversiteit Groningen. Papers published by the University ofGroningen, have stated that a 60 mg dose of pure micronized colistinsulfomethate could be effectively delivered as an inhalable dry powderutilizing this technology.

In preferred embodiments, the aerosol formulation is delivered as a drypowder using a dry powder inhaler wherein the particles emitted from theinhaler have an MMAD in the range of about 1 μm □ to about 5 μm and aGSD about less than 2.

Examples of suitable dry powder inhalers and dry powder dispersiondevices for use in the delivery of compounds and compositions accordingto the present invention include but are not limited to those disclosedin U.S. Pat. No. 7,520,278; U.S. Pat. No. 7,322,354; U.S. Pat. No.7,246,617; U.S. Pat. No. 7,231,920; U.S. Pat. No. 7,219,665; U.S. Pat.No. 7,207,330; U.S. Pat. No. 6,880,555; U.S. Pat. No. 5,522,385; U.S.Pat. No. 6,845,772; U.S. Pat. No. 6,637,431; U.S. Pat. No. 6,329,034;U.S. Pat. No. 5,458,135; U.S. Pat. No. 4,805,811; and U.S. PublishedPatent Application No. 2006/0237010.

In one embodiment, the pharmaceutical formulation according to theinvention is a dry powder for inhalation which is formulated fordelivery by a Diskus®-type device. The Diskus® device comprises anelongate strip formed from a base sheet having a plurality of recessesspaced along its length and a lid sheet hermetically but peelably sealedthereto to define a plurality of containers, each container havingtherein an inhalable formulation containing a predetermined amount ofactive ingredient either alone or in admixture with one or more carriersor excipients (e.g., lactose) and/or other therapeutically activeagents. Preferably, the strip is sufficiently flexible to be wound intoa roll. The lid sheet and base sheet will preferably have leading endportions which are not sealed to one another and at least one of theleading end portions is constructed to be attached to a winding means.Also, preferably the hermetic seal between the base and lid sheetsextends over their whole width. To prepare the dose for inhalation, thelid sheet may preferably be peeled from the base sheet in a longitudinaldirection from a first end of the base sheet.

In one embodiment, the pharmaceutical formulation according to theinvention is a dry powder for inhalation which is formulated fordelivery using a single-dose disposable inhaler, and particularly theTwincer™ inhaler. The Twincer™ inhaler comprises a foil laminate blisterwith one or more recesses and a lid sheet hermetically but peelablysealed thereto to define a plurality of containers. Each container hastherein an inhalable formulation containing a predetermined amount ofactive ingredient(s) either alone or in admixture with one or morecarriers or excipeints (e.g., lactose). The lid sheet will preferablyhave a leading end portion which is constructed to project from the bodyof the inhaler. The patient would operate the device and therebyadminister the aerosol formulation by 1) removing the outer packagingoverwrap, 2) pulling the foil tab to uncover the drug in the blister and3) inhaling the drug from the blister.

In another embodiment, the pharmaceutical formulation according to theinvention is a dry powder for inhalation wherein the dry powder isformulated into microparticles as described in PCT Publication No.WO2009/015286 or WO2007/114881, both to NexBio. Such microparticles aregenerally formed by adding a counterion to a solution containing acompound of the invention in a solvent, adding an antisolvent to thesolution; and gradually cooling the solution to a temperature belowabout 25° C., to form a composition containing microparticles comprisingthe compound. The microparticles comprising the compound may then beseparated from the solution by any suitable means such as sedimentation,filtration or lyophillization. Suitable counterions, solvents andantisolvents for preparing microparticles of the compounds of theinvention are described in WO2009/015286.

In another embodiment, a pharmaceutical composition according to theinvention is delivered as a dry powder using a metered dose inhaler.Non-limiting examples of metered dose inhalers and devices include thosedisclosed in U.S. Pat. No. 5,261,538; U.S. Pat. No. 5,544,647; U.S. Pat.No. 5,622,163; U.S. Pat. No. 4,955,371; U.S. Pat. No. 3,565,070; U.S.Pat. No. 3,361,306 and U.S. Pat. No. 6,116,234 and U.S. Pat. No.7,108,159. In a preferred embodiment, a compound of the invention isdelivered as a dry powder using a metered dose inhaler wherein theemitted particles have an MMAD that is in the range of about 1 μm toabout 5 μm and a GSD that is less than about 2 μm.

Liquid aerosol formulations for delivery to the endobronchial space orlung by inhalation may for example be formulated as aqueous solutions orsuspensions or as aerosols delivered from pressurized packs, such asmetered dose inhalers, with the use of suitable liquefied propellants,softmist inhalers, or nebulizers. Such aerosol compositions suitable forinhalation can be either a suspension or a solution and generallycontain the active ingredient(s) together with a pharmaceuticallyacceptable carrier or diluent (e.g., water (distilled or sterile),saline, hypertonic saline, or ethanol) and optionally one or more othertherapeutically active agents.

Aerosol compositions for delivery by pressurized metered dose inhalerstypically further comprise a pharmaceutically acceptable propellant.Examples of such propellants include fluorocarbon or hydrogen-containingchlorofluorocarbon or mixtures thereof, particularly hydrofluoroalkanes,e.g., dichlorodifluoromethane, trichlorofluoromethane,dichlorotetrafluoroethane, especially 1,1,1,2-tetrafluoroethane,1,1,1,2,3,3,3,-heptafluoro-n-propane or a mixture thereof. The aerosolcomposition may be excipient free or may optionally contain additionalformulation excipients well known in the art such as surfactants e.g.,oleic acid or lecithin and cosolvents e.g., ethanol. Pressurizedformulations will generally be retained in a canister (e.g., an aluminumcanister) closed with a valve (e.g., a metering valve) and fitted intoan actuator provided with a mouthpiece.

In another embodiment, a pharmaceutical composition according to theinvention is delivered as a liquid using a metered dose inhaler.Non-limiting examples of metered dose inhalers and devices include thosedisclosed in U.S. Pat. Nos. 6,253,762, 6,413,497, 7,601,336, 7,481,995,6,743,413, and 7,105,152. In a preferred embodiment, a compound of theinvention is delivered as a dry powder using a metered dose inhalerwherein the emitted particles have an MMAD that is in the range of about1 μm to about 5 μm and a GSD that is less than about 2.

In one embodiment the aerosol formulation is suitable for aerosolizationby a jet nebulizer, or ultrasonic nebulizer including static andvibrating porous plate nebulizers. Liquid aerosol formulations fornebulization may be generated by solubilizing or reconstituting a solidparticle formulation or may be formulated with an aqueous vehicle withthe addition of agents such as acid or alkali, buffer salts, andisotonicity adjusting agents. They may be sterilized by in-processtechniques such as filtration, or terminal processes such as heating inan autoclave or gamma irradiation. They may also be presented innon-sterile form.

Patients can be sensitive to the pH, osmolality, and ionic content of anebulized solution. Therefore these parameters should be adjusted to becompatible with the active ingredient and tolerable to patients. Themost preferred solution or suspension of active ingredient will containa chloride concentration >30 mM at pH 4.5-7.4, preferably 5.0-5.5, andan osmolality of from about 800-1600 mOsm/kg. The pH of the solution canbe controlled by either titration with common acids (hydrochloric acidor sulfuric acid, for example) or bases (sodium hydroxide, for example)or via the use of buffers. Commonly used buffers include citratebuffers, acetate buffers, and phosphate buffers. Buffer strengths canrange from 2 mM to 50 mM.

Such formulations may be administered using commercially availablenebulizers or other atomizer that can break the formulation intoparticles or droplets suitable for deposition in the respiratory tract.Non-limiting examples of nebulizers which may be employed for theaerosol delivery of a composition of the invention include pneumatic jetnebulizers, vented or breath enhanced jet nebulizers, or ultrasonicnebulizers including static or vibrating porous plate nebulizers.

A jet nebulizer utilizes a high velocity stream of air blasting upthrough a column of water to generate droplets. Particles unsuitable forinhalation impact on walls or aerodynamic baffles. A vented or breathenhanced nebulizer works in essentially the same way as a jet nebulizerexcept that inhaled air passes through the primary droplet generationarea to increase the output rate of the nebulizer while the patientinhales.

In an ultrasonic nebulizer, vibration of a piezoelectric crystal createssurface instabilities in the drug reservoir that cause droplets to beformed. In porous plate nebulizers pressure fields generated by sonicenergy force liquid through the mesh pores where it breaks into dropletsby Rayleigh breakup. The sonic energy may be supplied by a vibratinghorn or plate driven by a piezoelectric crystal, or by the mesh itselfvibrating. Non-limiting examples of atomizers include any single or twinfluid atomizer or nozzle that produces droplets of an appropriate size.A single fluid atomizer works by forcing a liquid through one or moreholes, where the jet of liquid breaks up into droplets. Twin fluidatomizers work by either forcing both a gas and liquid through one ormore holes, or by impinging a jet of liquid against another jet ofeither liquid or gas.

The choice of nebulizer which aerosolizes the aerosol formulation isimportant in the administration of the active ingredient(s). Differentnebulizers have differing efficiencies based their design and operationprinciple and are sensitive to the physical and chemical properties ofthe formulation. For example, two formulations with different surfacetensions may have different particle size distributions. Additionally,formulation properties such as pH, osmolality, and permeant ion contentcan affect tolerability of the medication, so preferred embodimentsconform to certain ranges of these properties.

In a preferred embodiment, the formulation for nebulization is deliveredto the endobronchial space as an aerosol having an MMAD between about 1μm and about 5 μm and a GSD less than 2 using an appropriate nebulizer.To be optimally effective and to avoid upper respiratory and systemicside effects, the aerosol should not have a MMAD greater than about 5 μmand should not have a GSD greater than about 2. If an aerosol has anMMAD larger than about 5 μm or a GSD greater than about 2 μm. a largepercentage of the dose may be deposited in the upper airways decreasingthe amount of drug delivered to the desired site in the lowerrespiratory tract. If the MMAD of the aerosol is smaller than about 1 mOthen a large percentage of the particles may remain suspended in theinhaled air and may then be exhaled during expiration.

The compounds of the invention may also be administered bytransbronchoscopic lavage.

In another aspect, the invention provides a method of promotinghydration of mucosal surfaces or restoring mucosal defense in a human inneed thereof, comprising administering to the human a pharmaceuticalcomposition comprising a compound of the invention, wherein saidcompound is administered in an effective amount. In one preferredembodiment, the method comprises administering the pharmaceuticalcomposition as an inhalable composition comprising an amount of acompound of the invention that is sufficient to achieve dissolvedconcentration of the compound on the airway surfaces of from about 10⁻⁹,10⁻⁸, or 10⁻⁷ to about 10⁻⁴, 10⁻³, 10⁻², or 10⁻¹ Moles/liter, morepreferably from about 10⁻⁹ to about 10⁻⁴ Moles/liter.

In another aspect, the invention provides a method of treating any oneof: a disease associated with reversible or irreversible airwayobstruction, chronic obstructive pulmonary disease (COPD), asthma,bronchiectasis (including bronchiectasis due to conditions other thancystic fibrosis), acute bronchitis, chronic bronchitis, post-viralcough, cystic fibrosis, emphysema, pneumonia, panbronchiolitis,transplant-associate bronchiolitis, and ventilator-associatedtracheobronchitis or preventing ventilator-associated pneumonia in ahuman in need thereof, comprising administering to the human apharmaceutical composition comprising a compound of the invention,wherein said compound is administered in an effective amount. In onepreferred embodiment, the method comprises administering thepharmaceutical composition as an inhalable composition comprising anamount of a compound of the invention that is sufficient to achievedissolved concentration of the compound on the airway surfaces of fromabout 10⁻⁹, 10⁻⁸, or 10⁻⁷ to about 10⁻⁴, 10⁻³, 10⁻², or 10⁻¹Moles/liter, more preferably from about 10⁻⁹ to about 10⁻⁴ Moles/liter.

In another aspect, the invention provides a method of treating any oneof dry mouth (xerostomia), dry skin, vaginal dryness, sinusitis,rhinosinusitis, or nasal dehydration, including nasal dehydrationbrought on by administering dry oxygen, dry eye, Sjogren'sdisease-associated dry eye, promoting ocular or corneal hydration,treating distal intestinal obstruction syndrome, treating otitis media,primary ciliary diskinesia, distal intestinal obstruction syndrome,esophagitis, constipation, or chronic diverticulitis in a human in needthereof, comprising administering to the human a pharmaceuticalcomposition comprising a compound of the invention, wherein saidcompound is administered in an effective amount.

Preferred unit dosage formulations for the compounds of the inventionare those containing an effective amount of the active ingredient or anappropriate fraction thereof.

It should be understood that in addition to the ingredients particularlymentioned above, the formulations of this invention may include otheragents conventional in the art having regard to the type of formulationin question for example those suitable for oral administration mayinclude flavoring agents.

The compositions of the present invention may be formulated forimmediate, controlled or sustained release as desired for the particularcondition being treated and the desired route of administration. Forexample, a controlled release formulation for oral administration may bedesired for the treatment of constipation in order to maximize deliveryof the active agent to colon. Such formulations and suitable excipientsfor the same are well known in the art of pharmacy. Because the freebase of the compound is generally less soluble in aqueous solutions thanthe salt, compositions comprising a free base of a compound of Formula Imay be employed to provide more sustained release of active agentdelivered by inhalation to the lungs. An active agent present in thelungs in particulate form which has not dissolved into solution is notavailable to induce a physiological response, but serves as a depot ofbioavailable drug which gradually dissolves into solution. As anotherexample, a formulation may employ both a free base and salt form of acompound of the invention to provide both immediate release andsustained release of the active ingredient for dissolution into themucus secretions of, for example, the nose.

ENaC blockers described in this invention can be administered by topicaladministration to the eyes of a patient in need of such treatment. ENaCblockers of Formula I are administered to the ocular surface of asubject, in an amount effective to reduce dry eye symptoms and toimprove hydration of the tear film. Preferably, ENaC blockers areadministered as a liquid or gel suspension in the form of drops, sprayor gel. Alternatively, ENaC blockers can be applied to the eye vialiposomes. ENaC blockers can also be contained within, carried by, orattached to contact lenses, punctual plugs or other compatiblecontrolled release materials, which are placed on the eye. ENaC blockerscan also be contained within a swab or sponge which can be applied tothe ocular surface. ENaC blockers can also be contained within a liquidspray which can be applied to the ocular surface. Another embodiment ofthe present invention involves an injection of ENaC blockers directlyinto the lacrimal tissues or onto the eye surface.

The topical solution containing ENaC blockers can contain aphysiologically compatible vehicle, as those skilled in the ophthalmicart can select using conventional criteria. The ophthalmic vehiclesinclude, but are not limited to, saline solution, water polyethers suchas polyethylene glycol, polyvinyls such as polyvinyl alcohol andpovidone, cellulose derivatives such as methylcellulose andhydroxypropyl methylcellulose, polycarbophil, petroleum derivatives suchas mineral oil and white petrolatum, animal fats such as lanolin,polymers of acrylic acid such as carboxypolymethylene gel, vegetablefats such as peanut oil and polysaccharides such as dextrans, andglycosaminoglycans such as sodium hyaluronate and salts such as sodiumchloride and potassium chloride.

The topical formulation optionally includes a preservative, such asbenzalkonium chloride and other inactive ingredients such as EDTA. ThepH of the formulation is adjusted by adding any physiologically andophthamologically acceptable pH adjusting acids, bases or buffers towithin the range of about 4.5 to 7.5; preferably 5 to 7. Examples ofacids include acetic, boric, citric, lactic, phosphoric, hydrochloric,and the like, and examples of bases include sodium hydroxide, sodiumphosphate, sodium borate, sodium citrate, sodium acetate, sodiumlactate, tromethamine, THAM (trishydroxymethylamino-methane), and thelike. Salts and buffers include citrate/dextrose, sodium bicarbonate,ammonium chloride and mixtures of the aforementioned acids and bases.

The osmotic pressure of the topical formulation of ENaC blockers isgenerally from about 200 to about 400 milliosmolar (mOsM), morepreferably from 260 to 340 mOsM. The osmotic pressure can be adjusted byusing appropriate amounts of physiologically and ophthamologicallyacceptable ionic or non-ionic agents. Sodium chloride is a preferredionic agent, and the amount of sodium chloride ranges from about 0.01%to about 1% (w/v), and preferably from about 0.05% to about 0.85% (w/v).Equivalent amounts of one or more salts made up of cations such aspotassium, ammonium and the like and anions such as chloride, citrate,ascorbate, borate, phosphate, bicarbonate, sulfate, thiosulfate,bisulfate, sodium bisulfate, ammonium sulfate, and the like can be usedin addition to or instead of sodium chloride to achieve osmolalitywithin the above-stated range. Further, non-ionic agents such asmannitol, dextrose, sorbitol, glucose and the like can also be used toadjust the osmolality.

The concentration of ENaC blockers included in the topical formulationis an amount sufficient to reduce dry eye symptoms and/or improvehydration of the tear film. This formulation is preferably an aqueoussolution of ENaC blockers and is in the range of 0.0001-0.3%, preferably0.001% to 0.1%, more preferably 0.003-0.05%, and most preferably about0.03% (w/v). “About” as used herein, refers to ±15% of the recitedvalue. The formulation optionally includes a preservative, such asbenzalkonium chloride (0.003% w/v) and inactive ingredients: edetatesodium, purified water, sodium chloride, sodium phosphate monobasic,sodium hydroxide, and/or hydrochloric acid to adjust the pH to about4-8.

The daily topical dose to reduce dry eye symptoms and improve tear filmcomposition can be divided among one or several unit doseadministrations. The total daily dose for ENaC blockers, for example,can range from one drop (about 50 μl), one to four times a day,depending upon the age and condition of the subject. A preferred regimenfor ENaC blockers is one drop of 0.03% (w/v) solution, about one to twotimes a day.

Liquid pharmaceutical compositions of ENaC blockers for producing eyedrops can be prepared by combining ENaC blockers with a suitablevehicle, such as sterile pyrogen free water or sterile saline bytechniques known to those skilled in the art.

Combinations

The compounds of the invention may be formulated and/or used incombination with other therapeutically active agents. Examples of othertherapeutically active agents which may be formulated or used incombination with the compounds of the invention include but are notlimited to osmolytes, anti-inflammatory agents, anticholinergic agents,β-agonists (including selective β₂-agonists), P2Y2 receptor agonists,peroxisome proliferator-activated receptor (PPAR) delta agonists, otherepithelial sodium channel blockers (ENaC receptor blockers), cysticfibrosis transmembrane conductance regulator (CFTR) modulators, kinaseinhibitors, antiinfective agents, antihistamines, non-antibioticanti-inflammatory macrolides, elastase and protease inhibitors, andmucus or mucin modifying agents, such as surfactants.

The present invention thus provides, as another aspect, a compositioncomprising an effective amount of a compound of the invention and one ormore other therapeutically active agents selected from osmolytes,anti-inflammatory agents, anticholinergic agents, β-agonists (includingselective β₂-agonists), P2Y2 receptor agonists, PPAR delta agonists,ENaC receptor blockers, cystic fibrosis transmembrane conductanceregulator (CFTR) modulators, kinase inhibitors, antiinfective agents,antihistamines, non-antibiotic anti-inflammatory macrolides, elastaseand protease inhibitors, and mucus or mucin modifying agents, such assurfactants. Use of the compounds of the invention in combination withone or more other therapeutically active agents (particularly osmolytes)may lower the dose of the compound of the invention that is required tosufficiently hydrate mucosal surfaces, thereby reducing the potentialfor undesired side-effects attributable to systemic blocking of sodiumchannels such as for example in the kidneys.

“Osmolytes” according to the present invention are molecules orcompounds that are osmotically active. “Osmotically active” moleculesand compounds are membrane-impermeable (i.e., essentiallynon-absorbable) on the airway or pulmonary epithelial surface. The terms“airway surface” and “pulmonary surface,” as used herein, includepulmonary airway surfaces such as the bronchi and bronchioles, alveolarsurfaces, and nasal and sinus surfaces. Suitable osmolytes include ionicosmolytes (i.e., salts), and non-ionic osmolytes (i.e., sugars, sugaralcohols, and organic osmolytes). In general, osmolytes (both ionic andnon-ionic) used in combination with the compounds of the invention arepreferably osmolytes that do not promote, or in fact deter or retardbacterial growth. Osmolytes suitable for use in the present inventionmay be in racemic form or in the form of an enantiomer, diastereomer,tautomer, polymorph or pseudopolymorph.

Examples of ionic osmolytes useful in the present invention include anysalt of a pharmaceutically acceptable anion and a pharmaceuticallyacceptable cation. Preferably, either (or both) of the anion and cationare osmotically active and not subject to rapid active transport, inrelation to the airway surfaces to which they are administered. Suchcompounds include but are not limited to anions and cations that arecontained in FDA approved commercially marketed salts, see, e.g.,Remington: The Science and Practice of Pharmacy, Vol. II, pg. 1457(19^(th) Ed. 1995), and can be used in any combination as known in theart.

Specific examples of pharmaceutically acceptable osmotically activeanions include but are not limited to, acetate, benzenesulfonate,benzoate, bicarbonate, bitartrate, bromide, calcium edetate, camsylate(camphorsulfonate), carbonate, chloride, citrate, dihydrochloride,edetate, edisylate (1,2-ethanedisulfonate), estolate (lauryl sulfate),esylate (1,2-ethanedisulfonate), fumarate, gluceptate, gluconate,glutamate, glycollylarsanilate (p-glycollamidophenylarsonate),hexylresorcinate, hydrabamine (N,N′-Di(dehydroabietyl)ethylenediamine),hydrobromide, hydrochloride, hydroxynaphthoate, iodide, isethionate,lactate, lactobionate, malate, maleate, mandelate, mesylate,methylbromide, methylnitrate, methylsulfate, mucate, napsylate, nitrate,nitrite, pamoate (embonate), pantothenate, phosphate or diphosphate,polygalacturonate, salicylate, stearate, subacetate, succinate, sulfate,tannate, tartrate, teoclate (8-chlorotheophyllinate), triethiodide,bicarbonate, etc. Preferred anions include chloride, sulfate, nitrate,gluconate, iodide, bicarbonate, bromide, and phosphate.

Specific examples of pharmaceutically acceptable osmotically activecations include but are not limited to, organic cations such asbenzathine (N,N′-dibenzylethylenediamine), chloroprocaine, choline,diethanolamine, ethylenediamine, meglumine (N-methyl D-glucamine),procaine, D-lysine, L-lysine, D-arginine, L-arginine, triethylammonium,N-methyl D-glycerol, and the like; and metallic cations such asaluminum, calcium, lithium, magnesium, potassium, sodium, zinc, iron,ammonium, and the like. Preferred organic cations include 3-carbon,4-carbon, 5-carbon and 6-carbon organic cations. Preferred cationsinclude sodium, potassium, choline, lithium, meglumine, D-lysine,ammonium, magnesium, and calcium.

Specific examples of ionic osmolytes that may be used in combinationwith a compound of the invention include but are not limited to, sodiumchloride (particularly hypertonic saline), potassium chloride, cholinechloride, choline iodide, lithium chloride, meglumine chloride, L-lysinechloride, D-lysine chloride, ammonium chloride, potassium sulfate,potassium nitrate, potassium gluconate, potassium iodide, ferricchloride, ferrous chloride, potassium bromide, and combinations of anytwo or more of the foregoing. In one embodiment, the present inventionprovides a combination of a compound of the invention and two differentosmotically active salts. When different salts are used, one of theanion or cation may be the same among the differing salts. Hypertonicsaline is a preferred ionic osmolyte for use in combination with thecompounds of the invention.

Non-ionic osmolytes include sugars, sugar-alcohols, and organicosmolytes. Sugars and sugar-alcohols useful as osmolytes in the presentinvention include but are not limited to 3-carbon sugars (e.g.,glycerol, dihydroxyacetone); 4-carbon sugars (e.g., both the D and Lforms of erythrose, threose, and erythrulose); 5-carbon sugars (e.g.,both the D and L forms of ribose, arabinose, xylose, lyxose, psicose,fructose, sorbose, and tagatose); and 6-carbon sugars (e.g., both the Dand L forms of altose, allose, glucose, mannose, gulose, idose,galactose, and talose, and the D and L forms of allo-heptulose,allo-hepulose, gluco-heptulose, manno-heptulose, gulo-heptulose,ido-heptulose, galacto-heptulose, talo-heptulose). Additional sugarsuseful in the practice of the present invention include raffinose,raffinose series oligosaccharides, and stachyose. Both the D and L formsof the reduced form of each sugar/sugar alcohol are also suitable forthe present invention. For example, glucose, when reduced, becomessorbitol; an osmolyte within the scope of the invention. Accordingly,sorbitol and other reduced forms of sugar/sugar alcohols (e.g.,mannitol, dulcitol, arabitol) are suitable osmolytes for use in thepresent invention. Mannitol is a preferred non-ionic osmolyte for use incombination with the compounds of the invention.

“Organic osmolytes” is generally used to refer to molecules that controlintracellular osmolality in the kidney. See e.g., J. S. Handler el al.,Comp. Biochem. Physiol, 117, 301-306 (1997); M. Burg, Am. J. Physiol.268, F983-F996 (1995). Organic osmolytes include but are not limited tothree major classes of compounds: polyols (polyhydric alcohols),methylamines, and amino acids. Suitable polyol organic osmolytes includebut are not limited to, inositol, myo-inositol, and sorbitol. Suitablemethylamine organic osmolytes include but are not limited to, choline,betaine, carnitine (L-, D- and DL forms), phosphorylcholine,lyso-phosphorylcholine, glycerophosphorylcholine, creatine, and creatinephosphate. Suitable amino acid organic osmolytes include but are notlimited to, the D- and L-forms of glycine, alanine, glutamine,glutamate, aspartate, proline and taurine. Additional organic osmolytessuitable for use in the present invention include tihulose andsarcosine. Mammalian organic osmolytes are preferred, with human organicosmolytes being most preferred. However, certain organic osmolytes areof bacterial, yeast, and marine animal origin, and these compounds mayalso be employed in the present invention.

Osmolyte precursors may be used in combination with the compounds of theinvention An “osmolyte precursor” as used herein refers to a compoundwhich is converted into an osmolyte by a metabolic step, eithercatabolic or anabolic. Examples of osmolyte precursors include but arenot limited to, glucose, glucose polymers, glycerol, choline,phosphatidylcholine, lyso-phosphatidylcholine and inorganic phosphates,which are precursors of polyols and methylamines. Precursors of aminoacid osmolytes include proteins, peptides, and polyamino acids, whichare hydrolyzed to yield osmolyte amino acids, and metabolic precursorswhich can be converted into osmolyte amino acids by a metabolic stepsuch as transamination. For example, a precursor of the amino acidglutamine is poly-L-glutamine, and a precursor of glutamate ispoly-L-glutamic acid.

Chemically modified osmolytes or osmolyte precursors may also beemployed. Such chemical modifications involve linking the osmolyte (orprecursor) to an additional chemical group which alters or enhances theeffect of the osmolyte or osmolyte precursor (e.g., inhibits degradationof the osmolyte molecule). Such chemical modifications have beenutilized with drugs or prodrugs and are known in the art. (See, forexample, U.S. Pat. Nos. 4,479,932 and 4,540,564; Shek, E. et al., J.Med. Chem. 19:113-117 (1976); Bodor, N. et al., J. Pharm. Sci.67:1045-1050 (1978); Bodor, N. et al., J. Med. Chem. 26:313-318 (1983);Bodor, N. et al., J. Pharm. Sci. 75:29-35 (1986).

Preferred osmolytes for use in combination with the compounds of theinvention include sodium chloride, particular hypertonic saline, andmannitol.

For the formulation of 7% and >7% hypertonic saline, formulationscontaining bicarbonate anions may be particularly useful, especially forrespiratory disorders with cystic fibrosis transmembrane conductanceregulator (CFTR) dysfunction such as CF or COPD. Recent findingsindicate that, although the relative ratio of HCO₃ conductance/Cl⁻conductance is between 0.1 and 0.2 for single CFTR channels activatedwith cAMP and ATP, the ratio in the sweat duct can range from virtually0 to almost 1.0, depending on conditions of stimulation. That is,combining cAMP+cGMP+α-ketoglutarate can yield CFTR HCO₃ ⁻ conductancealmost equal to that of Cl⁻ conductance (Quiton et al. Physiology, Vol.22, No. 3, 212-225, June 2007). Furthermore, formulations of 7% and >7%hypertonic saline containing bicarbonate anions may be particularlyuseful due to better control of the pH in the airway surface liquid.First, it has shown that that airway acidification occurs in CF (Tate etal. 2002) and that absent CFTR-dependent bicarbonate secretion can leadto an impaired capacity to respond to airway conditions associated withacidification of airway surface liquid layer (Coakley et al. 2003).Second, addition of HS solution without bicarbonate to the surface ofthe lung may further dilute the bicarbonate concentrations, andpotentially reduce the pH or the ability to respond to airwayacidification within the airway surface liquid layer. Therefore additionof bicarbonate anions to HS may help maintain or improve the pH ofairway surface liquid layer in CF patients.

Due to this evidence, inclusion of bicarbonate anion in the formulationof 7% or >7% hypertonic saline administered by the method of thisinvention would be particularly useful. Formulations containing up to 30to 200 mM concentrations of bicarbonate anions are of particularinterest for 7% or >7% HS solutions.

Hypertonic saline is understood to have a salt concentration greaterthan that of normal saline, i.e. greater than 9 g/L or 0.9% w/v, andhypotonic saline has a salt concentration less than that of normalsaline. Hypotonic saline solutions useful in the formulations andmethods of treatment herein may have a salt concentration from about 1%to about 23.4% (w/v). In one embodiment the hypertonic saline solutionhas a salt concentration from about 60 g/L (6% w/v) to about 100 g/L(10% w/v). In another embodiment, the saline solution has a saltconcentration from about 70 g/L (7% w/v) to about 100 g/L (10% w/v). Infurther embodiments, the saline solution has salt concentrations of a)from about 0.5 g/L (0.05% w/v) to about 70 g/L (7% w/v); b) from about 1g/L (0.1% w/v) to about 60 g/L (6% w/v); c) from about 1 g/L (0.1% w/v)to about 50 g/L (5% w/v); d) from about 1 g/L (0.1% w/v) to about 40 g/L(4% w/v); e) from about 1 g/L (0.1% w/v) to about 30 g/L (3% w/v); andf) from about 1 g/L (0.1% w/v) to about 20 g/L (2% w/v).

Specific concentrations of saline solutions useful in the formulationsand methods of treatment herein include, independently, those havingsalt concentrations of 1 g/L (0.1% w/v), 2 g/L (0.2% w/v), 3 g/L (0.3%w/v), 4 g/L (0.4% w/v), 5 g/L (0.5% w/v), 6 g/L (0.6% w/v), 7 g/L (0.7%w/v), 8 g/L (0.8% w/v), 9 g/L (0.9% w/v), 10 g/L (1% w/v), 20 g/L (2%w/v), 30 g/L (3% w/v), 40 g/L (4% w/v), 50 g/L (5% w/v), 60 g/L (6%w/v), 70 g/L (7% w/v), 80 g/L (8% w/v), 90 g/L (9% w/v), 100 g/L (10%w/v), 110 g/L (11% w/v), 120 g/L (12% w/v), 130 g/L (13% w/v), 140 g/L(14% w/v), and 150 g/L (15% w/v). Saline concentrations between each ofthese listed concentrations/percentages may also be used, such as salineof 1.7 g/L (0.17% w/v), 28 g/L (2.8% w/v), 35 g/L (3.5% w/v), and 45 g/L(4.5% w/v). Each of the ranges and specific concentrations of saline maybe used with the formulations, methods of treatment, regimens, and kitsdescribed herein.

Also intended within the scope of this invention are chemically modifiedosmolytes or osmolyte precursors. Such chemical modifications involvelinking to the osmolyte (or precursor) an additional chemical groupwhich alters or enhances the effect of the osmolyte or osmolyteprecursor (e.g., inhibits degradation of the osmolyte molecule). Suchchemical modifications have been utilized with drugs or prodrugs and areknown in the art. (See, for example, U.S. Pat. Nos. 4,479,932 and4,540,564; Shek, E. et al., J. Med. Chem. 19:113-117 (1976); Bodor, N.et al., J. Pharm. Sci. 67:1045-1050 (1978); Bodor, N. et al., J. Med.Chem. 26:313-318 (1983); Bodor, N. et al., J. Pharm. Sci. 75:29-35(1986), each incorporated herein by reference.

Suitable anti-inflammatory agents for use in combination with thecompounds of the invention include corticosteroids and non-steroidalanti-inflammatory drugs (NSAIDs), particularly phosphodiesterase (PDE)inhibitors. Examples of corticosteroids for use in the present inventioninclude oral or inhaled corticosteroids or prodrugs thereof. Specificexamples include but are not limited to ciclesonide,desisobutyryl-ciclesonide, budesonide, flunisolide, mometasone andesters thereof (e.g., mometasone furoate), fluticasone propionate,fluticasone furoate, beclomethasone, methyl prednisolone, prednisolone,dexamethasone,6α,9α-difluoro-17α-[(2-furanylcarbonyl)oxy]-11-hydroxy-16α-methyl-3-oxo-androsta-1,4-diene-17β-carbothioicacid S-fluoromethyl ester,6α,9α-difluoro-11β-hydroxy-16α-methyl-3-oxo-17α-propionyloxy-androsta-1,4-diene-17β-carbothioicacid S-(2-oxo-tetrahydro-furan-3S-yl) ester, beclomethasone esters(e.g., the 17-propionate ester or the 17,21-dipropionate ester,fluoromethyl ester, triamcinolone acetonide, rofleponide, or anycombination or subset thereof. Preferred corticosteroids for formulationor use in combination with the compounds of the invention are selectedfrom ciclesonide, desisobutyryl-ciclesonide, budesonide, mometasone,fluticasone propionate, and fluticasone furoate, or any combination orsubset thereof.

NSAIDs for use in the present invention include but are not limited tosodium cromoglycate, nedocromil sodium, phosphodiesterase (PDE)inhibitors (e.g., theophylline, aminophylline, PDE4 inhibitors, mixedPDE3/PDE4 inhibitors or mixed PDE4/PDE7 inhibitors), leukotrieneantagonists, inhibitors of leukotriene synthesis (e.g., 5 LO and FLAPinhibitors), nitric oxide synthase (iNOS) inhibitors, proteaseinhibitors (e.g., tryptase inhibitors, neutrophil elastase inhibitors,and metalloprotease inhibitors) β2-integrin antagonists and adenosinereceptor agonists or antagonists (e.g., adenosine 2a agonists), cytokineantagonists (e.g., chemokine antagonists) or inhibitors of cytokinesynthesis (e.g., prostaglandin D2 (CRTh2) receptor antagonists).Examples of leukotriene modifiers suitable for administration by themethod of this invention include monteleukast, zileuton and zafirlukast.

The PDE4 inhibitor, mixed PDE3/PDE4 inhibitor or mixed PDE4/PDE7inhibitor may be any compound that is known to inhibit the PDE4 enzymeor which is discovered to act as a PDE4 inhibitor, and which areselective PDE4 inhibitors (i.e., compounds which do not appreciablyinhibit other members of the PDE family). Examples of specific PDE4inhibitors for formulation and use in combination with the compounds ofthe present invention include but are not limited to roflumilast,pumafentrine, arofylline, cilomilast, tofimilast, oglemilast,tolafentrine, piclamilast, ibudilast, apremilast,2-[4-[6,7-diethoxy-2,3-bis(hydroxymethyl)-1-naphthalenyl]-2-pyridinyl]-4-(3-pyridinyl)-1(2H)-phthalazinone(T2585),N-(3,5-dichloro-4-pyridinyl)-1-[(4-fluorophenyl)methyl]-5-hydroxy-α-oxo-1H-indole-3-acetamide(AWD-12-281,4-[(2R)-2-[3-(cyclopentyloxy)-4-methoxyphenyl]-2-phenylethyl]-pyridine(CDP-840),2-[4-[[[[2-(1,3-benzodioxol-5-yloxy)-3-pyridinyl]carbonyl]amino]methyl]-3-fluorophenoxy]-(2R)-propanoicacid (CP-671305),N-(4,6-dimethyl-2-pyrimidinyl)-4-[4,5,6,7-tetrahydro-2-(4-methoxy-3-methylphenyl)-5-(4-methyl-1-piperazinyl)-1H-indol-1-yl]-benzenesulfonamide,(2E)-2-butenedioate (YM-393059),9-[(2-fluorophenyl)methyl]-N-methyl-2-(trifluoromethyl)-9H-purin-6-amine(NCS-613), N-(2,5-dichloro-3-pyridinyl)-8-methoxy-5-quinolinecarboxamide(D-4418),N-[(3R)-9-amino-3,4,6,7-tetrahydro-4-oxo-1-phenylpyrrolo[3,2,1-][1,4]benzodiazepin-3-yl]-3H-purin-6-amine(PD-168787),3-[[3-(cyclopentyloxy)-4-methoxyphenyl]methyl]-N-ethyl-8-(1-methylethyl)-3H-purin-6-aminehydrochloride (V-11294A),N-(3,5-dichloro-1-oxido-4-pyridinyl)-8-methoxy-2-(trifluoromethyl)-5-quinolinecarboxamide(Sch351591),5-[3-(cyclopentyloxy)-4-methoxyphenyl]-3-[(3-methylphenyl)methyl]-(3S,5S)-2-piperidinone(HT-0712),5-(2-((1R,4R)-4-amino-1-(3-(cyclopenyloxy)-4-methyoxyphenyl)cyclohexyl)ethynyl)-pyrimidine-2-amine,cis-[4-cyano-4-(3-cyclopropylmethoxy-4-difluoromethoxyphenyl)cyclohexan-1-ol], and4-[6,7-diethoxy-2,3-bis(hydroxymethyl)-1-naphthalenyl]-1-(2-methoxyethyl)-2(1H)-pyridinone(T-440), and any combination or subset thereof.

Leukotriene antagonists and inhibitors of leukotriene synthesis includezafirlukast, montelukast sodium, zileuton, and pranlukast.

Anticholinergic agents for formulation or use in combination with thecompounds of the invention include but are not limited to muscarinicreceptor antagonists, particularly including pan antagonists andantagonists of the M₃ receptors. Exemplary compounds include thealkaloids of the belladonna plants, such as atropine, scopolamine,homatropine, hyoscyamine, and the various forms including salts thereof(e.g., anhydrous atropine atropine sulfate, atropine oxide or HCl,methylatropine nitrate, homatropine hydrobromide, homatropine methylbromide, hyoscyamine hydrobromide, hyoscyamine sulfate, scopolaminehydrobromide, scopolamine methyl bromide), or any combination or subsetthereof.

Additional anticholinergics for formulation and use in combination withthe methantheline, propantheline bromide, anisotropine methyl bromide orValpin 50, aclidinium bromide, glycopyrrolate (Robinul), isopropamideiodide, mepenzolate bromide, tridihexethyl chloride, hexocycliummethylsulfate, cyclopentolate HCl, tropicamide, trihexyphenidyl CCl,pirenzepine, telenzepine, and methoctramine, or any combination orsubset thereof.

Preferred anticholinergics for formulation and use in combination withthe compounds of the invention include ipratropium (bromide), oxitropium(bromide) and tiotropium (bromide), or any combination or subsetthereof.

Examples of β-agonists for formulation and use in combination with thecompounds of the invention include but are not limited to salmeterol,R-salmeterol, and xinafoate salts thereof, albuterol or R-albuterol(free base or sulfate), levalbuterol, salbutamol, formoterol (fumarate),fenoterol, procaterol, pirbuterol, metaprterenol, terbutaline and saltsthereof, and any combination or subset thereof.

P2Y2 receptor agonists for formulation and use in combination with thecompounds of the invention may be employed in an amount effective tostimulate chloride and water secretion by airway surfaces, particularlynasal airway surfaces. Suitable P2Y2 receptor agonists are known in theart and are described for example, in columns 9-10 of U.S. Pat. No.6,264,975, and also U.S. Pat. Nos. 5,656,256 and 5,292,498.

P2Y₂ agonists that can be administered by the methods of this inventioninclude P2Y₂ receptor agonists such as ATP, UTP, UTP-.gamma.-S anddinucleotide P2Y₂ receptor agonists (e.g. denufosol or diquafosol) or apharmaceutically acceptable salt thereof. The P2Y₂ receptor agonist istypically included in an amount effective to stimulate chloride andwater secretion by airway surfaces, particularly nasal airway surfaces.Suitable P2Y₂ receptor agonists are described in, but are not limitedto, U.S. Pat. No. 6,264,975, U.S. Pat. No. 5,656,256, U.S. Pat. No.5,292,498, U.S. Pat. No. 6,348,589, U.S. Pat. No. 6,818,629, U.S. Pat.No. 6,977,246, U.S. Pat. No. 7,223,744, U.S. Pat. No. 7,531,525 and U.S.Pat. AP. 2009/0306009 each of which is incorporated herein by reference.

Combination therapies and formulations herein can include adenosine 2b(A2b) agonists, also, including BAY 60-6583, NECA(N-ethylcarboxamidoadenosine), (S)-PHPNECA, LUF-5835 and LUF-5845. A2bagonists that may be used are described by Volpini et al., Journal ofMedicinal Chemistry 45 (15): 3271-9 (2002); Volpini et al., CurrentPharmaceutical Design 8 (26): 2285-98 (2002); Baraldi et al., Journal ofMedicinal Chemistry 47 (6): Cacciari et al., 1434-47 (2004); MiniReviews in Medicinal Chemistry 5 (12): 1053-60 (December 2005); Baraldiet al., Current Medicinal Chemistry 13 (28): 3467-82 (2006); Beukers etal., Medicinal Research Reviews 26 (5): 667-98 (September 2006); Elzeinet al., Bioorganic & Medicinal Chemistry Letters 16 (2): 302-6 (January2006); Carotti, et al., Journal of Medicinal Chemistry 49 (1): 282-99(January 2006); Tabrizi et al., Bioorganic & Medicinal Chemistry 16 (5):2419-30 (March 2008); and Stefanachi, et al., Bioorganic & MedicinalChemistry 16 (6): 2852-69 (March 2008).

Examples of other ENaC receptor blockers for formulation and use incombination with the compounds of the invention include but are notlimited to amiloride and derivatives thereof such as those compoundsdescribed in U.S. Pat. No. 6,858,615, and PCT Publication Nos.WO2003/070182, WO2004/073629, WO2005/018644, WO2006/022935,WO2007/018640, and WO2007/146869, all to Parion Sciences, Inc.

Small molecule ENaC blockers are capable of directly preventing sodiumtransport through the ENaC channel pore. ENaC blocker that can beadministered in the combinations herein include, but are not limited to,amiloride, benzamil, phenamil, and amiloride analogues as exemplified byU.S. Pat. No. 6,858,614, U.S. Pat. No. 6,858,615, U.S. Pat. No.6,903,105, U.S. Pat. No. 6,995,160, U.S. Pat. No. 7,026,325, U.S. Pat.No. 7,030,117, U.S. Pat. No. 7,064,129, U.S. Pat. No. 7,186,833, U.S.Pat. No. 7,189,719, U.S. Pat. No. 7,192,958, U.S. Pat. No. 7,192,959,U.S. Pat. No. 7,241,766, U.S. Pat. No. 7,247,636, U.S. Pat. No.7,247,637, U.S. Pat. No. 7,317,013, U.S. Pat. No. 7,332,496, U.S. Pat.No. 7,345,044, U.S. Pat. No. 7,368,447, U.S. Pat. No. 7,368,450, U.S.Pat. No. 7,368,451, U.S. Pat. No. 7,375,107, U.S. Pat. No. 7,399,766,U.S. Pat. No. 7,410,968, U.S. Pat. No. 7,820,678, U.S. Pat. No.7,842,697, U.S. Pat. No. 7,868,010, U.S. Pat. No. 7,875,619.

ENaC proteolysis is well described to increase sodium transport throughENaC. Protease inhibitors block the activity of endogenous airwayproteases, thereby preventing ENaC cleavage and activation. Proteasesthat cleave ENaC include furin, meprin, matriptase, trypsin, channelassociated proteases (CAPs), and neutrophil elastases. Proteaseinhibitors that can inhibit the proteolytic activity of these proteasesthat can be administered in the combinations herein include, but are notlimited to, camostat, prostasin, furin, aprotinin, leupeptin, andtrypsin inhibitors.

Combinations herein may include one or more suitable nucleic acid (orpolynucleic acid), including but not limited to antisenseoligonucleotide, siRNA, miRNA, miRNA mimic, antagomir, ribozyme,aptamer, and decoy oligonucleotide nucleic acids. See, e.g., US PatentApplication Publication No. 20100316628. In general, such nucleic acidsmay be from 17 or 19 nucleotides in length, up to 23, 25 or 27nucleotides in length, or more. Examples include, but are not limitedto, those described in U.S. Pat. No. 7,517,865 and US PatentApplications Nos. 20100215588; 20100316628; 20110008366; and20110104255. In general, the siRNAs are from 17 or 19 nucleotides inlength, up to 23, 25 or 27 nucleotides in length, or more.

CFTR activity modulating compounds that can be administered in thecombinations of this invention include, but are not limited to,compounds described in US 2009/0246137 A1, US 2009/0253736 A1, US2010/0227888 A1, U.S. Pat. No. 7,645,789, US 2009/0246820 A1, US2009/0221597 A1, US 2010/0184739 A1, US 2010/0130547 A1, US 2010/0168094A1 and issued U.S. Pat. No. 7,553,855; U.S. Pat. No. 7,772,259 B2, U.S.Pat. No. 7,405,233 B2, US 2009/0203752, U.S. Pat. No. 7,499,570.

Mucus or mucin modifying agents useful in the combinations and methodsherein include reducing agents, surfactants and detergents,expectorants, and deoxyribonuclease agents.

Mucin proteins are organized into high molecular weight polymers via theformation of covalent (disulfide) and non-covalent bonds. Disruption ofthe covalent bonds with reducing agents is a well-established method toreduce the viscoelastic properties of mucus in vitro and is predicted tominimize mucus adhesiveness and improve clearance in vivo. Reducingagents are well known to decrease mucus viscosity in vitro and commonlyused as an aid to processing sputum samples⁸. Examples of reducingagents include sulfide containing molecules or phosphines capable ofreducing protein di-sulfide bonds including, but not limited to,N-acetyl cysteine, N-acystelyn, carbocysteine, glutathione,dithiothreitol, thioredoxin containing proteins, andtris(2-carboxyethyl)phosphine.

N-acetyl cysteine (NAC) is approved for use in conjunction with chestphysiotherapy to loosen viscid or thickened airway mucus^({12}).Clinical studies evaluating the effects of oral or inhaled NAC in CF andCOPD have reported improvements in the rheologic properties of mucus andtrends toward improvements in lung function and decreases in pulmonaryexacerbations⁹. However, the preponderance of clinical data suggeststhat NAC is at best a marginally effective therapeutic agent fortreating airway mucus obstruction when administered orally or byinhalation. A recent Cochrane review of the existing clinical literatureon the use of NAC found no evidence to support the efficacy of NAC forCF¹⁰. The marginal clinical benefit of NAC reflects:

NAC is a relative inefficient reducing agent which is only partiallyactive on the airway surface. Very high concentrations of NAC (200 mM or3.26%) are required to fully reduce Muc5B, a major gel-forming airwaymucin, in vitro. Furthermore, in the pH environment of the airwaysurface (measured in the range of pH 6.0 to 7.2 in CF and COPDairways)¹¹, NAC exists only partially in its reactive state as anegatively charge thiolate. Thus, in the clinic, NAC is administered atvery high concentrations. However, it is predicted that current aerosoldevices will not be able to achieve therapeutic concentrations of even a20% Mucomyst solution on distal airway surfaces within the relativelyshort time domains (7.5-15 minutes) typically used.

In non-clinical studies, ¹⁴C-labeled NAC, administered by inhalation,exhibits rapid elimination from the lungs with a half-life ranging from6 to 36 minutes¹²

NAC is administered as a highly concentrated, hypertonic inhalationsolution (20% or 1.22 molar) and has been reported to causebronchoconstriction and cough. In many cases, it is recommended that NACbe administered with a bronchodilator to improve the tolerability ofthis agent.

Thus, reducing agents such as NAC are not well suited for bolus aerosoladministration. However, it is anticipated that delivery of reducingagents by pulmonary aerosol infusion would increase the effectiveness,while allowing for a decrease in the concentration of reducing agent inthe inhalation solution (predicted to increase tolerability).

Surfactants and detergents are spreading agents shown to decrease mucusviscoelasticity, improving mucus clearability. Examples of surfactantsinclude DPPC, PF, palmitic acid, palmitoyl-oleoylphosphatidylglycerol,surfactant proteins (e.g. SP-A, B, or C), or may be animal derived (e.g.from cow or calf lung lavage or extracted from minced pig lung) orcombinations thereof. See, e.g., U.S. Pat. Nos. 7,897,577; 5,876,970;5,614,216; 5,100,806; and 4,312,860. Examples of surfactant productsinclude Exosurf, Pumactant, KL-4, Venticute, Alveofact, Curosurf,Infasurf, and Survanta. Examples of detergents include, but are notlimited to, Tween-80 and triton-X 100.

Any suitable expectorant can be used, including but not limited toguaifenesin (see, e.g., U.S. Pat. No. 7,345,051). Any suitabledeoxyribonuclease can be used, including but not limited to DornaseAlpha. (see, e.g., U.S. Pat. No. 7,482,024).

Examples of kinase inhibitors include inhibitors of NFkB, PI3K(phosphatidylinositol 3-kinase), p38-MAP kinase and Rho kinase.

Antiinfective agents for formulation and use in combination with thecompounds of the invention include antivirals and antibiotics. Examplesof suitable antivirals include Tamiflu® and Relenza®. Examples ofsuitable antibiotics include but are not limited to aztreonam (arginineor lysine), fosfomycin, and aminoglycosides such as tobramycin, or anycombination or subset thereof. Additional antiinfective agents that maybe used herein include aminoglycosides, Daptomycin, Fluoroquinolones,Ketolides, Carbapenems, Cephalosporins, Erythromycin, Linezolid,Penicillins, Azithromycin, Clindamycin, Oxazolidinones, Tetracyclines,and Vancomycin.

Examples of useful carbapenam antibiotics are impenam, panipenam,meropenam, biapenam, MK-826, DA-1131, ER-35786, lenapenam, S-4661,CS-834 (prodrug of R-95867), KR-21056 (prodrug of KR-21012), L-084(prodrug of LJC 11036) and CXA-101.

Antihistamines (i.e., H1-receptor antagonists) for formulation and usein combination with the compounds of the invention include but are notlimited to: ethanolamines such as diphenhydramine HCl, carbinoxaminemaleate, doxylamine, clemastine fumarate, diphenylhydramine HCl anddimenhydrinate; ethylenediamines such as pyrilamine maleate(metpyramine), tripelennamine HCl, tripelennamine citrate, andantazoline; alkylamines such as pheniramine, chloropheniramine,bromopheniramine, dexchlorpheniramine, triprolidine and acrivastine;pyridines such as methapyrilene, piperazines such as hydroxyzine HCl,hydroxyzine pamoate, cyclizine HCl, cyclizine lactate, meclizine HCl andcetirizine HCl; piperidines such as astemisole, levocabastine HCl,loratadine, descarboethoxy loratadine, terfenadine, and fexofenadineHCl; tri- and tetracyclics such as promethazine, chlorpromethazinetrimeprazine and azatadine; and azelastine HCl, or any combination orsubset thereof.

Examples of other classes of therapeutic agents suitable for use in thecombinations and methods herein include antivirals such as ribavirin,anti-fungal agents such as amphotericin, intraconazol and voriconazol,anti-rejection drugs such as cyclosporine, tacrolimus and sirolimus,immunomodulatory agents including steroids such as dexamethasone,anti-inflammatory agents including but not limited to cyclooxygenaseinhibitors, cytokine inhibitors, JAK inhibitors, and inhibitors ofT-cell function, bronchodilators including but not limited toanticholinergic agents such as atrovent, siRNAs, gene therapy vectors,aptamers, endothelin-receptor antagonists, alpha-1-antitrypsin andprostacyclins.

Examples of other classes of agents suitable for use in the combinationsand methods herein include viscosity enhancing or water retaining agentssuch as hyaluronic acid or carboxymethylcellulose, hormones includingestrogen or testosterone, and other agents used to treat dry eye diseaseincluding autologous serum and tear substitutes.

In the above-described methods of treatment and uses, a compound of theinvention may be employed alone, or in combination with one or moreother therapeutically active agents. Typically, any therapeuticallyactive agent that has a therapeutic effect in the disease or conditionbeing treated with the compound of the invention may be utilized incombination with the compounds of the invention, provided that theparticular therapeutically active agent is compatible with therapyemploying a compound of the invention. Typical therapeutically activeagents which are suitable for use in combination with the compounds ofthe invention include agents described above.

In one preferred embodiment, the compounds of the invention are used incombination with one or more osmolytes, particularly hypertonic salineor mannitol.

In another aspect, the invention provides methods for treatment and usesas described above, which comprise administering an effective amount ofa compound of the invention and at least one other therapeuticallyactive agent. The compounds of the invention and at least one additionaltherapeutically active agent may be employed in combinationconcomitantly or sequentially in any therapeutically appropriatecombination. The administration of a compound of the invention with oneor more other therapeutically active agents may be by administrationconcomitantly in 1) a unitary pharmaceutical composition, such as thecompositions described above, or 2) separate pharmaceutical compositionseach including one or more of the component active ingredients. Thecomponents of the combination may be administered separately in asequential manner wherein the compound of the invention is administeredfirst and the other therapeutically active agent is administered secondor vice versa.

In the embodiments wherein the compound of the invention is administeredin combination with one or more osmolytes, the administration of eachcomponent is preferably concomitant, and may be in a unitary compositionor separate compositions. In one embodiment, the compound of theinvention and one or more osmolytes are administered concomitantly bytransbrochoscopic lavage. In another embodiment, the compound of theinvention and one or more osmolytes are administered concomitantly byinhalation.

When a compound of the invention is used in combination with anothertherapeutically active agent, the dose of each compound may differ fromthat when the compound of the invention is used alone. Appropriate doseswill be readily determined by one of ordinary skill in the art. Theappropriate dose of the compound of the invention, the othertherapeutically active agent(s) and the relative timings ofadministration will be selected in order to achieve the desired combinedtherapeutic effect, and are within the expertise and discretion of theattendant physician, clinician or veterinarian.

The compounds of formula I-III may be synthesized according toprocedures known in the art. A representative synthetic procedure isshown in the scheme below:

These procedures are described in, for example, E. J. Cragoe, “TheSynthesis of Amiloride and Its Analogs” (Chapter 3) in Amiloride and ItsAnalogs, pp. 25-36, incorporated herein by reference. Other methods ofpreparing the compounds are described in, for example, U.S. Pat. No.3,313,813, incorporated herein by reference. See in particular MethodsA, B, C, and D described in U.S. Pat. No. 3,313,813. Additional methodsof preparing intermediates used in the preparation of compounds of theinstant invention are disclosed in U.S. Pat. No. 7,064,129, U.S. Pat.No. 6,858,615, U.S. Pat. No. 6,903,105, WO 2004/073629, WO 2007/146869,and WO 2007/018640, each of which is expressly incorporated byreference.

Several assays may be used to characterize the compounds of the presentinvention. Representative assays are discussed below.

Animal Models of Dry Eye Disease

(1) In Vitro Measure of Sodium Channel Blocking Activity

One assay used to assess mechanism of action and/or potency of thecompounds of the present invention involves the determination of lumenaldrug inhibition of airway epithelial sodium currents measured undershort circuit current (I_(SC)) using airway epithelial monolayersmounted in Ussing chambers. This assay is described in detail in Hirsh,A. J., Zhang, J., Zamurs, A., et al. Pharmacological properties ofN-(3,5-diamino-6-chloropyrazine-2-carbonyl)-N′-4-[4-(2,3-dihydroxypropoxy)phenyl]butyl-guanidinemethanesulfonate (552-02), a novel epithelial sodium channel blockerwith potential clinical efficacy for CF lung disease. J. Pharmacol. Exp.Ther. 2008; 325(1): 77-88.

Cells obtained from freshly excised human, dog, sheep or rodent airwaysare seeded onto porous 0.4 micron Snapwell™ Inserts (CoStar), culturedat air-liquid interface (ALI) conditions in hormonally defined media,and assayed for sodium transport activity (I_(SC)) while bathed in KrebsBicarbonate Ringer (KBR) in Using chambers. All test drug additions areto the lumenal bath with half-log dose addition protocols (from 1×10⁻¹¹M to 3×10⁻⁵ M), and the cumulative change in I_(SC) (inhibition)recorded. All drugs are prepared in dimethyl sulfoxide as stocksolutions at a concentration of 1×10⁻² M and stored at −20° C. Eightpreparations are typically run in parallel; two preparations per runincorporate amiloride and/or benzamil as positive controls. After themaximal concentration (5×10⁻⁵ M) is administered, the lumenal bath isexchanged three times with fresh drug-free KBR solution, and theresultant I_(SC) measured after each wash for approximately 5 minutes induration. Reversibility is defined as the percent return to the baselinevalue for sodium current after the third wash. All data from the voltageclamps are collected via a computer interface and analyzed off-line.

Dose-effect relationships for all compounds are considered and analyzedby the Prism 3.0 program. IC₅₀ values, maximal effective concentrations,and reversibility are calculated and compared to amiloride and benzamilas positive controls. The potency of the sodium channel blockingactivity of representative compounds relative to amiloride in freshlyexcised cells from canine airways is shown in Table 1.

TABLE 1 Potency of sodium channel blocking activity of Formula Icompounds. Potency of Sodium Channel Blockade Compound In Canine Cells(IC₅₀) Amiloride 781.0 30 33.1 35 13.8 45 2.1 15 4.6 42 24.6  9 3.2 516.6 59 41.3 145  7.3 82 4.6

(2) Pharmacological Effects of Compounds on Tear Volume in an AnimalModel of Dry Eye Disease In Vivo.

The effects of compounds on tear volume was assessed in a rat model ofdry eye disease in which Sprague Dawley rats undergo surgical lacrimalgland excision (ExLac Model) to reduce normal tear volume. The lacrimalexcision reduces normal tear volume by ˜50% (Table 2).

Both the ipsilateral and contralateral eyes were dosed with 5 μl of testarticle solution. Tear production was measured using the ZoneQuickcotton thread with impregnated phenol red dye. The folded end of thethread was held in the lateral-ventral conjunctival cul-de-sac for 10seconds. The length of tear wicking onto the thread was determined bymeasuring the length of the thread that changes color from yellow tored. Use of a stereomicroscope was assist in the accurate measurement(recorded in millimeters) of the wicking/color change. Tear volume wasassessed pre-dose, and 15, 30, 60, 120, and 360 minute post-dose. Thechange in ocular hydration produced by representative compounds in ExLacrats relative to amiloride is shown in Table 2 and FIGS. 1-16. Forreference, the effect of a saline vehicle is shown for ExLac rats andnormal (no lacrimal excision surgery) rats.

TABLE 2 Ocular hydrating activity of Formula I componds. Compound RatModel 6 h Ocular Hydration (AUC₀₋₆) Vehicle ExLac 24.2 Amiloride ExLac30.4 51 ExLac 38.0 75 ExLac 38.2 59 ExLac 39.7 46 ExLac 39.9 116 ExLac40.9 45 ExLac 42.3 102 ExLac 43.2 145 ExLac 44.7 133 ExLac 45.7 90 ExLac49.0 82 ExLac 49.8 15 ExLac 50.2 9 ExLac 50.2 42 ExLac 52.3 VehicleNormal 58.1

(3) Confocal Microscopy Assay of Amiloride Congener Transport

Virtually all amiloride-like molecules fluoresce in the ultravioletrange. This property of these molecules may be used to directly measurecellular update using confocal microscopy. Corneal cells were labeledusing calcein-AM dye by incubating with corneas for 45 minutes at 37 Cin DMEM media. Equimolar concentrations of 2 microliters of compound 9or amiloride were placed on the apical (epithelial) surface of mousecorneas for one hour at 37 C. Serial x-y images were obtained one hourpost-drug addition by confocal microscopy. The data shown in FIG. 16,show a x-z image of the corneas made up from the composite of the x-yimage stack. FIG. 16 shows that amiloride can fully penetrate the corneais one hour post-administration, but Compound 9 remains associated withthe apical (epithelial) surface.

(4) In Vitro Drug Metabolism

The metabolic stability of 9 and 15 was assessed in plasma (rat, rabbit,dog and human) and hepatocytes (rat and dog). Compounds were addedeither directly to plasma or to hepatocyte suspensions at a finalconcentration of 2.5 or 10 μM, respectively, and incubated at 37° C. forup to six hours. Aliquots were removed at various time points andquenched. The amount of parent compound was quantified viaUPLC-fluorescence analysis. The amount of parent compound remaining wascalculated based on the total peak area at the time of sampling dividedby the total peak area at initiation. The results presented in Tables 3and 4 show that 9 was stable towards metabolic hydrolysis in both plasmaand hepatocytes among the species evaluated, whereas, 15 was rapidlymetabolized in both plasma and hepatocytes. These results confirm thatthe 15 with amide linkages in the naturally occurring S configuration issusceptible to enzymatic hydrolysis, whereas, the amide linkages in theR configuration are stable towards hydrolysis.

TABLE 3 Plasma Stability of Compounds Matrix Compound 9 Compound 15 Rat87% 10% Rabbit 89% 8.7%  Dog 103% 18% Human 101% 7.6% 

TABLE 4 Hepatocyte Stability of Compounds Matrix Compound 9 Compound 15Compound 45 Rat 100% 14% 83% Dog 94% 19% 75%

(5) In Vitro Assays of Compound Metabolism

Airway epithelial cells have the capacity to metabolize drugs during theprocess of transepithelial absorption. Further, although less likely, itis possible that drugs can be metabolized on airway epithelial surfacesby specific ectoenzyme activities. Perhaps more likely as anecto-surface event, compounds may be metabolized by the infectedsecretions that occupy the airway lumens of patients with lung disease,e.g. cystic fibrosis. Thus, a series of assays is performed tocharacterize the compound metabolism that results from the interactionof test compounds with human airway epithelia and/or human airwayepithelial lumenal products.

In the first series of assays, the interaction of test compounds in KBRas an “ASL” stimulant are applied to the apical surface of human airwayepithelial cells grown in the T-Col insert system. For most compounds,metabolism (generation of new species) is tested for using highperformance liquid chromatography (HPLC) to resolve chemical species andthe endogenous fluorescence properties of these compounds to estimatethe relative quantities of test compound and novel metabolites. For atypical assay, a test solution (25 μl KBR, containing 10 μM testcompound) is placed on the epithelial lumenal surface. Sequential 5 to10 μl samples are obtained from the lumenal and serosal compartments forHPLC analysis of (1) the mass of test compound permeating from thelumenal to serosal bath and (2) the potential formation of metabolitesfrom the parent compound. In instances where the fluorescence propertiesof the test molecule are not adequate for such characterizations,radiolabeled compounds are used for these assays. From the HPLC data,the rate of disappearance and/or formation of novel metabolite compoundson the lumenal surface and the appearance of test compound and/or novelmetabolite in the basolateral solution is quantitated. The data relatingthe chromatographic mobility of potential novel metabolites withreference to the parent compound are also quantitated.

To analyze the potential metabolism of test compounds by CF sputum, a“representative” mixture of expectorated CF sputum obtained from 10 CFpatients (under IRB approval) has been collected. The sputum has been besolubilized in a 1:5 mixture of KBR solution with vigorous vortexing,following which the mixture was split into a “neat” sputum aliquot andan aliquot subjected to ultracentrifugation so that a “supernatant”aliquot was obtained (neat-cellular; supernatant=liquid phase). Typicalstudies of compound metabolism by CF sputum involve the addition ofknown masses of test compound to “neat” CF sputum and aliquots of CFsputum “supernatant” incubated at 37 OC, followed by sequential samplingof aliquots from each sputum type for characterization of compoundstability/metabolism by HPLC analysis as described above. As above,analysis of compound disappearance, rates of formation of novelmetabolities, and HPLC mobilities of novel metabolites are thenperformed.

(6) Pharmacological Effects and Mechanism of Action of the Drug inAnimals

The effect of compounds for enhancing mucociliary clearance (MCC) can bemeasured using an in vivo model described by Sabater et al., Journal ofApplied Physiology, 1999, pp. 2191-2196, incorporated herein byreference.

Methods Animal Preparation:

Adult ewes (ranging in weight from 25 to 35 kg) were restrained in anupright position in a specialized body harness adapted to a modifiedshopping cart. The animals=heads were immobilized and local anesthesiaof the nasal passage was induced with 2% lidocaine. The animals werethen nasally intubated with a 7.5 mm internal diameter endotracheal tube(ETT). The cuff of the ETT was placed just below the vocal cords and itsposition was verified with a flexible bronchoscope. After intubation theanimals were allowed to equilibrate for approximately 20 minutes priorto initiating measurements of mucociliary clearance.

Administration of Radio-Aerosol:

Aerosols of ^(99m)Tc-Human serum albumin (3.1 mg/ml; containingapproximately 20 mCi) were generated using a Raindrop Nebulizer whichproduces a droplet with a median aerodynamic diameter of 3.6 μm. Thenebulizer was connected to a dosimetry system consisting of a solenoidvalve and a source of compressed air (20 psi). The output of thenebulizer was directed into a plastic T connector; one end of which wasconnected to the endotracheal tube, the other was connected to a pistonrespirator. The system was activated for one second at the onset of therespirator's inspiratory cycle. The respirator was set at a tidal volumeof 500 mL, an inspiratory to expiratory ratio of 1:1, and at a rate of20 breaths per minute to maximize the central airway deposition. Thesheep breathed the radio-labeled aerosol for 5 minutes. A gamma camerawas used to measure the clearance of ^(99m)Tc-Human serum albumin fromthe airways. The camera was positioned above the animal's back with thesheep in a natural upright position supported in a cart so that thefield of image was perpendicular to the animal's spinal cord. Externalradio-labeled markers were placed on the sheep to ensure properalignment under the gamma camera. All images were stored in a computerintegrated with the gamma camera. A region of interest was traced overthe image corresponding to the right lung of the sheep and the countswere recorded. The counts were corrected for decay and expressed aspercentage of radioactivity present in the initial baseline image. Theleft lung was excluded from the analysis because its outlines aresuperimposed over the stomach and counts can be swallowed and enter thestomach as radio-labeled mucus.Treatment Protocol (Assessment of Activity at t-Zero):

A baseline deposition image was obtained immediately after radio-aerosoladministration. At time zero, after acquisition of the baseline image,vehicle control (distilled water), positive control (amiloride), orexperimental compounds were aerosolized from a 4 ml volume using a PariLC JetPlus nebulizer to free-breathing animals. The nebulizer was drivenby compressed air with a flow of 8 liters per minute. The time todeliver the solution was 10 to 12 minutes. Animals were extubatedimmediately following delivery of the total dose in order to preventfalse elevations in counts caused by aspiration of excess radio-tracerfrom the ETT. Serial images of the lung were obtained at 15-minuteintervals during the first 2 hours after dosing and hourly for the next6 hours after dosing for a total observation period of 8 hours. Awashout period of at least 7 days separated dosing sessions withdifferent experimental agents.

Treatment Protocol (Assessment of Activity at t-4 hours):The following variation of the standard protocol was used to assess thedurability of response following a single exposure to vehicle control(distilled water), positive control compounds (amiloride or benzamil),or investigational agents. At time zero, vehicle control (distilledwater), positive control (amiloride), or investigational compounds wereaerosolized from a 4 ml volume using a Pari LC JetPlus nebulizer tofree-breathing animals. The nebulizer was driven by compressed air witha flow of 8 liters per minute. The time to deliver the solution was 10to 12 minutes. Animals were restrained in an upright position in aspecialized body harness for 4 hours. At the end of the 4-hour periodanimals received a single dose of aerosolized ^(99m)Tc-Human serumalbumin (3.1 mg/ml; containing approximately 20 mCi) from a RaindropNebulizer. Animals were extubated immediately following delivery of thetotal dose of radio-tracer. A baseline deposition image was obtainedimmediately after radio-aerosol administration. Serial images of thelung were obtained at 15-minute intervals during the first 2 hours afteradministration of the radio-tracer (representing hours 4 through 6 afterdrug administration) and hourly for the next 2 hours after dosing for atotal observation period of 4 hours. A washout period of at least 7 daysseparated dosing sessions with different experimental agents.Statistics: Data were analyzed using SYSTAT for Windows, version 5. Datawere analyzed using a two-way repeated ANOVA (to assess overalleffects), followed by a paried t-test to identify differences betweenspecific pairs. Significance was accepted when P was less than or equalto 0.05. Slope values (calculated from data collected during the initial45 minutes after dosing in the t-zero assessment) for mean MCC curveswere calculated using linear least square regression to assessdifferences in the initial rates during the rapid clearance phase.

Examples

Having generally described this invention, a further understanding canbe obtained by reference to certain specific examples which are providedherein for purposes of illustration only and are not intended to belimiting unless otherwise specified.

Preparation of Sodium Channel Blockers

Materials and methods. The present invention also provides processes forpreparing the compounds of the invention and to the syntheticintermediates useful in such processes, as described in detail below.

Certain abbreviations and acronyms are used in describing the syntheticprocesses and experimental details. Although most of these would beunderstood by one skilled in the art, the following table contains alist of many of these abbreviations and acronyms.

Abbreviation Meaning AcOH Acetic Acid AIBN Azobisisobutyrolnitrile DIADDiisopropyl azidocarboxylate DIPEA N,N-Diisopropylethylamine DCEdichloroethane DCM dichloromethane DMF dimethylformamide Et Ethyl EtOAcor EA ethyl acetate EtOH Ethanol ESI electrospray ionization HATU2-(1H-7-Azabenzotriazol-1-yl)-1,1,3,3-tetramethyl uroniumhexafluorophosphate HPLC High performance liquid chromatography iPrOHIsopropyl alcohol i.t. or IT intratracheal Me Methyl MeOH methanol m/zor m/e mass to charge ratio MH⁺ mass plus 1 MH⁻ mass minus 1 MIC minimalinhibitory concentration MS or ms mass spectrum rt or r.t. roomtemperature R_(f) Retardation factor t-Bu tert-butyl THF tetrahydrofuranTLC or tlc thin layer chromatography δ parts per million down field fromtetramethylsilane Cbz Benzyloxycarbonyl, i.e. —(CO)O-benzyl AUC Areaunder the curve or peak MTBE Methyl tertiary butyl ether t_(R) Retentiontime GC-MS Gas chromatography-mass spectrometry wt % Percent by weight hHours min Minutes MHz megahertz TFA Trifluoroacetic acid UV UltravioletBoc tert-butyloxycarbonyl DIAD Diisopropyl azodicarboxylate AcOH AceticAcid DIPEA N,N-Diisopropylethylamine or Hünig's base Ph₃PTriphenylphosine

The compounds of Formula I may be synthesized using techniques known inthe art. A representative synthetic procedure is illustrated in Scheme 1below.

Preparation of(R)-6-(2,3-bis(tert-butoxycarbonyl)guanidino)-2-((tertbutoxycarbonyl)amino)hexanoicacid (3)

To a solution of N-α-Boc-D-lysine (13.0 g, 52.7 mmol) in EtOH (290 mL)was added N,N′-bis-Boc-1-guanylpyrazole (16.3 g, 52.7 mmol) and triethylamine (10.6 g, 105 mmol). The reaction mixture was stirred at roomtemperature for 6 h. Solvent was removed and the residue was purified bycolumn chromatography (silica gel, 10:1 CH₂Cl₂/MeOH) to afford apyrazole salt (25.0 g) as colorless oil. The salt was dissolved in 1 NNaOH (300 mL) and neutralized with 1 N HCl (305 mL). The resultingprecipitate was filtered out and dried, to afford compound 3 (22.0 g,85%) as a white solid: ¹H NMR (400 MHz, CDCl₃) δ 11.48 (br s, 1H), 8.35(br s, 1H), 5.23 (d, J=7.5 Hz, 1H), 4.23 (br s, 1H), 3.48-3.25 (m, 2H),1.96-1.50 (m, 6H), 1.51 (s, 9H), 1.49 (s, 9H), 1.43 (s, 9H).

Preparation of Compound 5

To a solution of amino acid 3 (3.00 g, 6.14 mmol) in CH₂Cl₂ (100 mL) wasadded EEDQ (3.17 g, 12.8 mmol) and NMM (4.90 g, 49.1 mmol). The reactionmixture was stirred at room temperature for 10 min and then bis-amine 4(1.73 g, 3.07 mmol) was added. The resulting mixture was stirred at roomtemperature for 24 h. Amino acid 3 (900 mg, 1.84 mmol) was added and thereaction mixture was stirred for additional 16 h. Solvent was removedand the residue was purified by column chromatography (silica gel, 10:1CH₂Cl₂/EtOAc, 10:1 CH₂Cl₂/MeOH) to afford amide 5 (2.30 g, 57%) as acolorless solid: ¹H NMR (400 MHz, CD₃OD) δ 7.38-7.24 (m, 5H), 7.06 (d,J=8.4 Hz, 2H), 6.82 (d. J=8.4 Hz, 2H), 5.05 (s, 2H), 4.09-3.92 (m, 4H),3.68 (t, J=4.8 Hz, 4H), 3.58 (t, J=6.3 Hz, 1H), 3.28-3.21 (m, 4H),3.14-3.07 (m, 2H), 2.89-2.83 (m, 2H) 2.64-2.50 (m, 6H), 2.43 (br s, 4H),2.27 (s, 3H), 1.77-1.65 (m, 6H), 1.64-1.54 (m, 6H), 1.52 (s, 18H), 1.46(s, 18H), 1.42 (s, 18H).

Preparation of Compound 6

A suspension of compound 5 (2.30 g, 1.64 mmol) and 10% Pd/C (1.50 g) inEtOH (10 mL) was subjected to hydrogenation conditions (1 atm) for 4 hat room temperature. The reaction mixture was filtered through celiteand washed with EtOH. The filtrate was concentrated to afford an amine(2.10 g) as colorless oil. The crude was purified by columnchromatography (silica gel, 8:1 CH₂Cl₂/MeOH) to afford amine 6 (1.50 g,72%) as colorless oil: ¹H NMR (400 MHz, CD₃OD) δ 7.08 (d, J=8.5 Hz, 2H),6.83 (d, J=8.5 Hz, 2H), 4.15-3.88 (m, 4H), 3.28-3.21 (m, 8H), 2.84 (t,J=5.5 Hz, 2H), 2.78 (t, J=7.4 Hz, 2H), 2.58 (t, J=7.2 Hz, 6H), 1.84-1.52(m, 20H), 1.52 (s, 18H), 1.46 (s, 18H), 1.43 (s, 18H).

Preparation of Compound 8

To a solution of amine 6 (9.00 g, 7.12 mmol) and methyl3,5-diamino-6-chloropyrazine-2-carbonylcarbamimidothioate hydroiodicacid salt (7, 4.43 g, 11.3 mmol) in t-BuOH (90 mL) was added DIPEA (7.36g, 56.9 mmol) at room temperature. The reaction mixture was heated at70° C. in a sealed tube for 2 h, then cooled to room temperature, andconcentrated in vacuum. The residue was purified by columnchromatography (silica gel, 10:1 CH₂Cl₂/MeOH, 5:1:0.1 CHCl₃/MeOH/NH₄OH)to afford compound 8 (5.60 g, 53%) as a yellow solid: ¹H NMR (400 MHz,CD₃OD) δ 7.10 (d, J=8.7 Hz, 2H), 6.84 (d, J=8.7 Hz, 1H), 4.06-3.94 (m,4H), 3.29-3.20 (m, 6H), 2.87-2.80 (m, 2H), 2.64-2.53 (m, 6H), 1.78-1.64(m, 12H), 1.65-1.51 (m, 12H), 1.52 (s, 18H), 1.47 (s, 18H), 1.41 (s,18H).

Preparation of the hydrochloride salt of(2R,2′R)—N,N′-(3,3′-(2-(4-(4-(3-(3,5-diamino-6-chloropyrazine-2-carbonyl)guanidino)butyl)phenoxy)ethylazanediyl)bis(propane-3,1-diyl))bis(2-amino-6-guanidinohexanamide)compound 9

To a solution of compound 8 (5.60 g, 0.81 mmol) in EtOH (20 mL) wasadded 4 N aq HCl (120 mL) at room temperature and the reaction mixturewas stirred for 4 h at room temperature. The reaction mixture wasconcentrated in vacuum and the residue was purified by reverse phasecolumn chromatography and lyophilized to afford hydrochloric acid salt 9(1.5 g, 45%) as a yellow hygroscopic solid: ¹H NMR (400 MHz, D₂O) δ 7.22(d, J=8.2 Hz, 1H), 6.91 (d, J=8.2 Hz, 1H), 4.28 (br s, 2H), 3.89 (t,J=6.8 Hz, 2H), 3.60 (br s, 2H), 3.37-3.23 (m, 10H), 3.08 (t, J=7.2 Hz,4H), 2.59 (br s, 2H), 2.05-1.93 (m, 4H), 1.86-1.75 (m, 4H), 1.66 (br s,4H), 1.58-1.47 (m, 4H), 1.39-1.27 (m, 4H).

Preparation of(S)-6-(2,3-bis(tert-butoxycarbonyl)guanidino)-2-((tertbutoxycarbonyl)amino)hexanoicacid (11)

To a solution of N-α-Boc-L-lysine (1.00 g, 4.06 mmol) 16 in EtOH (30 mL)was added N,N′-bis-Boc-1-guanylpyrazole (1.36 g, 4.38 mmol) 2 andtriethyl amine (810 mg, 8.12 mmol). The reaction mixture was stirred atroom temperature for 6 h. Solvent was removed and the residue waspurified by column chromatography (silica gel, 10:1 CH₂Cl₂/MeOH) toafford a pyrazole salt (1.98 g) as colorless oil. The salt was dissolvedin 1 N NaOH (100 mL) and neutralized with 1 N HCl (105 mL). Theresulting precipitate was filtered out and dried, to afford compound 11(1.50 g, 76%) as a white solid: ¹H NMR (300 MHz, CDCl₃) δ 11.47 (br s,1H), 8.37 (br s, 1H), 5.22 (d, J=7.5 Hz, 1H), 4.26 (br s, 1H), 3.49-3.29(m, 2H), 1.98-1.51 (m, 6H), 1.50 (s, 9H), 1.49 (s, 9H), 1.44 (s, 9H).

Preparation of Compound 12

To a solution of amino acid 11 (6.00 g, 12.0 mmol) in CH₂Cl₂ (150 mL)was added EEDQ (5.00 g, 20.2 mmol) and NMM (10.0 g, 99.0 mmol). Thereaction mixture was stirred at room temperature for 10 min and thenbis-amine 4 (3.40 g, 6.00 mmol) was added. The resulting mixture wasstirred at room temperature for 48 h. Solvent was removed and theresidue was purified by column chromatography (silica gel, 10:1CH₂Cl₂/EtOAc, 10:1 CH₂Cl₂/MeOH) to afford amide 12 (4.98 g, 58%) as acolorless solid: ¹H NMR (300 MHz, CD₃OD) δ 7.33-7.31 (m, 5H), 7.06 (d,J=8.1 Hz, 1H), 6.82 (d, J=8.1 Hz, 1H), 5.05 (s, 2H), 4.10-4.01 (m, 4H),3.35-3.23 (m, 8H), 3.11 (t, J=6.9 Hz, 2H), 2.85 (t, J=5.4 Hz, 2H),2.44-2.41 (m, 6H), 1.72-1.51 (m, 20H), 1.51 (s, 18H), 1.46 (s, 18H),1.43 (s, 18H).

Preparation of Compound 13

A suspension of compound 12 (4.95 g, 3.54 mmol) and 10% Pd/C (2.50 g) inEtOH/AcOH (150 mL/5.0 mL) was subjected to hydrogenation conditions (1atm) for 16 h at room temperature. The reaction mixture was filteredthrough celite and washed with EtOH. The filtrate was concentrated toafford an acid salt (4.80 g) as colorless oil. The salt was neutralizedwith satd Na₂CO₃ and purified by column chromatography (silica gel, 8:1CH₂Cl₂/MeOH) to afford free base 13 (3.35 g, 75%) as colorless oil: ¹HNMR (300 MHz, CD₃OD) δ 7.08 (d. J=8.4 Hz, 1H), 6.82 (d, J=8.4 Hz, 1H),4.05-4.01 (m, 4H), 3.31-3.23 (m, 8H), 2.84 (t, J=5.4 Hz, 2H), 2.73 (t,J=6.9 Hz, 2H), 2.61-2.55 (m, 6H), 1.72-1.52 (m, 20H), 1.51 (s, 18H),1.46 (s, 18H), 1.43 (s, 18H).

Preparation of Compound 14

To a solution of amine 13 (3.30 g, 2.61 mmol) and methyl3,5-diamino-6-chloropyrazine-2-carbonylcarbamimidothioate hydroiodicacid salt (7, 1.62 g, 4.18 mmol) in t-BuOH (80 mL) was added DIPEA (2.70g, 20.8 mmol) at room temperature. The reaction mixture was heated at70° C. in a sealed tube for 2 h, then cooled to room temperature, andconcentrated in vacuum. The residue was purified by columnchromatography (silica gel, 10:1 CH₂Cl₂/MeOH, 5:1:0.1 CHCl₃/MeOH/NH₄OH)to afford compound 14 (1.78 g, 47%) as a yellow solid: ¹H NMR (300 MHz,CD₃OD) δ 7.10 (d, J=8.4 Hz, 1H), 6.85 (d, J=8.4 Hz, 1H), 4.05-3.99 (m,4H), 3.31-3.23 (m, 10H), 2.86 (t, J=5.4 Hz, 2H), 2.62-2.58 (m, 6H),1.70-1.52 (m, 20H), 1.51 (s, 18H), 1.48 (s, 18H), 1.46 (s, 18H).

Preparation of Compound 15

To a solution of compound 14 (1.20 g, 0.813 mmol) in EtOH (5 mL) wasadded 4 N aq HCl (25 mL) at room temperature and the reaction mixturewas stirred for 4 h at room temperature. The reaction mixture wasconcentrated in vacuum and the residue was purified by reverse phasecolumn chromatography and lyophilized to afford hydrochloric acid salt15 (356 mg, 40%) as a yellow hygroscopic solid: ¹H NMR (300 MHz, D₂O) δ7.12 (d, J=8.4 Hz, 1H), 6.81 (d, J=8.4 Hz, 1H), 4.18 (br s, 2H), 3.79(t, J=4.9 Hz, 2H), 3.50 (br s, 2H), 3.24-3.20 (m, 10H), 2.98 (t, J=6.9Hz, 2H), 2.50 (br s, 2H), 1.92-1.87 (m, 4H), 1.74-1.67 (m, 4H), 1.45 (brs, 4H), 1.28-1.21 (m, 4H).

General Description for the Preparation of the hydrochloride salt ofbenzyl 4-(4-(2-(bis(3-aminopropyl)amino)ethoxy)phenyl)butylcarbamate (4)

All non-aqueous reactions were carried out under an atmosphere of eithernitrogen or argon. Reagents and solvents were used as received fromsuppliers. Deionized water (DI water) was used for workups and toprepare diluted solutions. Thin-layer chromatography (TLC) was performedusing Merck silica-gel plates and visualized by UV light (254 nm) orappropriate stain. ¹H NMR and ¹³C NMR spectra were obtained on a BrukerAVANCE-400 Ultra Shield spectrometer at 400 MHz for proton and 100 MHzfor carbon, using CDCl₃, D₂O, or DMSO-d₆ as the solvents. The massspectra were obtained on an Agilent spectrometer using electrospray oratmospheric-pressure chemical ionization (APCI).

Step 1. Preparation of 5

A stirred solution of benzyl [4-(4-hydroxyphenyl)butyl]carbamate (17,500 g, 1670 mmol, 1.0 equiv,), tert-butyl(2-hydroxyethyl)carbamate (18,350.0 g, 2170 mmol, 1.3 equiv), and PPh₃ (568.0 g, 2170 mmol, 1.3 equiv,AVRA) in THF (7500 mL, 15 vol, Finar lot) was charged with DIAD (438.0g, 2170 mmol, 1.3 equiv, AVRA) dropwise at 0° C. over 30 min, andstirred at room temperature for 16 h. The progress of the reaction wasmonitored by TLC analysis (7:3 hexanes:EtOAc), which confirmed thepresence of ≈10% compound 17. 18 (81 g, 503 mmol, 0.3 equiv), PPh₃ (132g, 503 mmol, 0.3 equiv), and DIAD (102 g, 503 mmol, 0.3 equiv) wereadded at <10° C. and the mixture was stirred at room temperature for 16h. Having confirmed the complete consumption of compound 17, the solventwas evaporated under vacuum to afford crude 19 (2.50 kg, crude), whichwas used as produced in the next step.

Step 2. Preparation of 20

A stirred solution of 19 (2500 g) and HCl in dioxane (10,000 mL, Durga)was stirred at room temperature for 3-4 h. The progress of the reactionwas monitored by TLC (30% EtOAc: hexanes). After completion of thereaction, the solvent was evaporated under vacuum to 1/3 volume. Theresulting solid was triturated with MTBE (5000 mL, Savla Chemicals) andthe precipitate was filtered and dried under vacuum to afford 20 (370.0g, 58%) as a white solid.

Step 3. Preparation of 22 Preparation of 20 Free Base

Compound 20 (140.0 g) was dissolved in DI water (1500 mL) and the pH wasadjusted to ≈9 using solid Na₂CO₃ (Finar Reagents). The aqueous layerwas extracted with CH₂Cl₂ (3×500 mL, MSN lot). The combined organiclayers were dried over anhydrous Na₂SO₄ and evaporated under vacuum toafford the free base of 20 [75 g, 60%].

Reductive Amination

A stirred solution of 20 free base [75 g, 219 mmol, 1.0 equiv] and 21(95.0 g, 549 mmol, 2.5 equiv) in CH₂Cl₂(1500 mL, MSN) was charged withCH₃COOH (13.0 g, 219 mmol, 1.0 equiv, S.D. Fine-Chem) and stirred for 30min at room temperature, then cooled to 0-5° C. Na(OAc)₃BH (140.0 g, 660mmol, Aldrich lot) was added portionwise over 30 min, and the mixturewas stirred at room temperature for 16 h. The progress of the reactionwas monitored by TLC (9.5:0.5 CH₂Cl₂: MeOH, 2 runs). After the reactionwas complete, the reaction mixture was quenched with aqueous 1 N NaOHsolution, adjusting the pH to ≈9. The layers were separated and theaqueous layer was extracted with CH₂Cl₂ (2×500 mL, MSN). The combinedorganic layers were washed with water (1×300 mL), dried over anhydrousNa₂SO₄, and evaporated under vacuum to afford crude 22 (160.0 g) as athick, light green liquid. The crude was purified by columnchromatography (silica gel, 100-200 mesh, 4.9:0.1, CH₂Cl₂:MeOH aseluent, 2 purifications) to give pure 22 [61 g, 42%] as a pale yellowliquid.

Step 4. Preparation of 4

A mixture of 22 [130.0 g, 198 mmol] and HCl in IPA (≈20%, 650 mL, DurgaIndustries) was stirred for 3 h. The progress of the reaction wasmonitored by TLC (9.5:0.5, CH₂Cl₂:MeOH). After the completion of thereaction, the solvent was evaporated to 1/3 volume and MTBE (650 mL,Savla Chemicals) was added. A thick solid was precipitated; the solventwas decanted. The mixture was solvent-swapped with toluene (2×500 mL)and MTBE (2×1000 mL, Savla Chemicals) and dried under vacuum. Theresulting sticky solid was stirred in MTBE (1000 mL) for 1 h, thesolvent was decanted, and the product was dried under vacuum to afford 4(94.0 g, 84%, AMRI) as a highly hygroscopic, off-white solid.

Step 5. Preparation of 18

A stirred solution of 2-aminoethanol (200.0 g, 3274.3 mmol, 1.0 equiv)and TEA (497.0 g, 4911.4 mmol, 1.5 equiv) in CH₂Cl₂ (2400 mL, 12 vol,MSN) was charged with (Boc)₂O (856.0 g, 3926.1 mmol, 1.2 equiv, GlobeChemie) at 0-5° C., and was stirred at room temperature for 2 h,monitoring the progress of the reaction by TLC (9:1, CH₂Cl₂:MeOH). Afterthe complete consumption of the 2-aminoethanol, DI water (2500 mL) wasadded and the mixture was stirred for 10 min. The two layers wereseparated and the organic layer was washed with 0.2 N HCl (3000 mL) andDI water (1000 mL), dried over anhydrous Na₂SO₄, and evaporated undervacuum to afford 18 (482 g, 91%) as a pale green liquid.

Step 6. Preparation of 23

A stirred solution of 3-aminopropanol (250 g, 3334 mmol, 1.0 equiv, AlfaAesar) and TEA (505 g, 5000 mmol, 1.5 equiv, AVRA) in CH₂Cl₂ (3000 mL,12 vol, MSN 1) was charged with (Boc)₂O (872 g, 4000 mmol, 1.2 equiv,Globe Chemie lot) at 0-5° C., and was stirred at room temperature for 2h, monitoring the progress of the reaction by TLC (9:1, CH₂Cl₂:MeOH).After the completion of the reaction, water (3000 mL) was added and themixture was stirred for 10 min. The layers were separated and theorganic layer was washed with 0.2 N HCl (3000 mL) and DI water (1000mL), dried over anhydrous Na₂SO₄, and evaporated under vacuum to affordBoc-aminopropanol 23 (588 g, 100%,) as a pale green liquid.

Step 7. Preparation of 21

A stirred solution of 23 (100.0 g, 571 mmol, 1.0 equiv) in DMSO (600 mL,6 vol, Finar) was charged with IBX (243 g, 868 mmol, 1.5 equiv, QuiverTechnologies) portionwise over 30 min at room temperature, and wasstirred for 5 h. The progress of the reaction was monitored by TLC (9:1CH₂Cl₂: MeOH). After the completion of the reaction, the mixture wasdiluted with DI water (4000 mL). The solid was filtered and washed withDI water (1000 mL). The filtrate was extracted with ethyl acetate(2×1000 m, MSN). The combined organic layers were washed with saturatedNaHCO₃ (1×1000 mL) and DI water (1000 mL), dried over anhydrous Na₂SO₄,and evaporated under vacuum to afford 21 (71 g, 70%) as a yellow liquid.

Preparation of Compound 25

A solution of amino acid 24 (1.80 g, 3.94 mmol) in THF (50 mL) wascharged with DEPBT (1.23 g, 4.14 mmol) and DIPEA (1.27 g, 9.85 mmol).The reaction mixture was stirred at room temperature for 1 h andbis-amine 4 (900 mg, 1.97 mmol) was added. The resulting mixture wasstirred at room temperature for 16 h and 40° C. for 8 h. The solvent wasremoved and the residue was purified by column chromatography (silicagel, 5:1 CH₂Cl₂/EtOAc) to afford amide 3 (1.63 g, mixture with compound25 as a yellow solid, which was used directly in the next step.

Preparation of Compound 26

A solution of compound 25 (100 mg, mixture) in EtOH (3.0 mL) was chargedwith piperidine (1.0 mL). The reaction mixture was stirred at roomtemperature for 3 h. After the solvent was removed, the residue wasprecipitated from hexanes, washed with 1 N NaOH, and azeotroped withMeOH to afford compound 26 (40.0 mg, 36% over 2 steps) as a white solid:¹H NMR (300 MHz, CD₃OD) δ 7.32-7.27 (m, 5H), 7.06 (d, J=8.1 Hz, 2H),6.82 (d, J=8.1 Hz, 2H), 5.05 (s, 2H), 4.04 (t, J=5.4 Hz, 2H), 3.34-3.19(m, 5H), 3.11 (t, J=6.9 Hz, 2H), 3.04-2.98 (m, 5H), 2.85 (t, J=5.7 Hz,2H), 2.62-2.52 (m, 6H), 1.75-1.28 (m, 20H), 1.42 (s, 18H).

Preparation of Compound 27

A solution of compound 26 (300 mg, 0.329 mmol) in MeOH (10 mL) and water(5.0 mL) was charged with NaHCO₃ (56.0 mg, 0.666 mmol) and Boc₂O (56.0mg, 0.394 mmol). The reaction mixture was stirred for 4 h at roomtemperature. After the solvent was removed, the residue was washed withwater and azeotroped with MeOH to afford compound 27 (303 mg, 83%) as acolorless oil: ¹H NMR (300 MHz. CD₃OD) 7.33-7.29 (m, 5H), 7.06 (d, J=8.4Hz, 2H), 6.82 (d, J=8.4 Hz, 2H), 5.05 (s, 2H), 4.04 (t, J=5.1 Hz, 2H),3.94-3.93 (br s, 2H), 3.37-3.22 (m, 4H), 3.14-3.11 (m, 2H), 3.09-2.98(m, 4H), 2.90-2.86 (m, 2H), 2.61-2.52 (m, 6H), 1.69-1.29 (m, 20H), 1.42(s, 36H).

Preparation of Compound 28

A suspension of compound 27 (300 mg, 0.269 mmol) and 10% Pd/C (150 mg)in EtOH (4.0 mL) and AcOH (0.5 mL) was subjected to hydrogenationconditions (1 atm) for 4 h at room temperature. The reaction mixture wasfiltered through celite and washed with EtOH. The filtrate wasconcentrated and washed with MTBE to afford compound 28 (285 mg, 96%) asa colorless oil: ¹H NMR (300 MHz, CD₃OD) δ 7.12 (d, J=8.4 Hz, 2H), 6.87(d, J=8.4 Hz, 2H), 4.12 (br s, 2H), 3.93 (br s, 2H), 3.40-3.30 (m, 4H),3.07-2.91 (m, 8H), 2.78-2.61 (m, 6H), 1.93 (s, 6H), 1.77-1.42 (m, 20H),1.42 (s, 36H).

Preparation of Compound 29

A solution of compound 28 (280 mg, 0.213 mmol) and methyl3,5-diamino-6-chloropyrazine-2-carbonylcarbamimidothioate hydroiodicacid salt (7, 132 mg, 0.339 mmol) in EtOH (5.0 mL) was charged withDIPEA (220 mg, 1.70 mmol) at room temperature. The reaction mixture washeated at 70° C. for 2 h, cooled to room temperature, and concentratedin vacuum. The residue was purified by column chromatography (silicagel, 10:1 CH₂Cl₂/MeOH, 5:1:0.1 CHCl₃/MeOH/NH₄OH) to afford compound 29(189 mg, 63%) as a yellow solid: ¹H NMR (400 MHz, CD₃OD) δ 7.10 (d,J=8.4 Hz, 2H), 6.84 (d, J=8.4 Hz, 2H), 4.03 (t, J=5.6 Hz, 2H), 3.95 (brs, 2H), 3.34-3.29 (m, 6H), 3.00 (t, J=6.8 Hz, 4H), 2.84 (br s, 2H),2.61-2.56 (m, 6H), 1.70-1.42 (m, 20H), 1.42 (s, 36H).

Preparation of the Hydrochloride Salt of(2S,2′S)—N,N′-(3,3′-(2-(4-(4-(3-(3,5-diamino-6-chloropyrazine-2-carbonyl)guanidino)butyl)phenoxy)ethylazanediyl)bis(propane-3,1-diyl))bis(2,6-diaminohexanamide)(Compound 30)

A solution of compound 29 (188 mg, 0.157 mmol) in EtOH (2.0 mL) wascharged with 4 N aqueous HCl (6.0 mL) at room temperature and thereaction mixture was stirred for 4 h at room temperature. The reactionmixture was concentrated in vacuum and the residue was recrystallizedfrom EtOH/H₂O and lyophilized to afford hydrochloric acid salt 30 (140mg, 87%) as a yellow hygroscopic solid: ¹H NMR (400 MHz, D₂O) δ 7.22 (d,J=8.4 Hz, 2H), 6.91 (d, J=8.4 Hz, 2H), 4.28 (br s, 2H), 3.90 (t, J=6.8Hz, 2H), 3.61 (br s, 2H), 3.36-3.23 (m, 10H), 2.94 (t, J=7.6 Hz, 4H),2.59 (br s, 2H), 1.98-1.87 (m, 4H), 1.85-1.82 (m, 4H), 1.66-1.62 (m,8H), 1.40-1.38 (m, 4H).

Preparation of Compound 32

A solution of compound 26 (180 mg, 0.197 mmol) in MeOH (5.0 mL) wascharged with compound 31 (132 mg, 0.493 mmol) and AcOH (60 mg, 0.985mmol). The reaction mixture was stirred at room temperature for 20 minand NaCNBH₃ (57.3 mg, 0.788 mmol) was added. After the reaction mixturewas stirred at room temperature for 16 h, the solvent was removed invacuum. The residue was washed with saturated Na₂CO₃, azeotroped withMeOH, and purified by column chromatography (silica gel, 10:1CH₂Cl₂/MeOH, 5:1:0.1 CHCl₃/MeOH/NH₄OH) to afford compound 32 (183 mg,mixture) as a colorless oil, which was used directly in the next step.

Preparation of Compound 33

A suspension of compound 32 (180 mg, 0.127 mmol) and 10% Pd/C (100 mg)in EtOH (5.0 mL) and AcOH (1.0 mL) was subjected to hydrogenationconditions (1 atm) for 36 h at room temperature. The reaction mixturewas filtered through celite and washed with EtOH. The filtrate wasconcentrated and washed with MTBE to afford compound 33 (129 mg, 46%over 2 steps) as a colorless oil: ¹H NMR (300 MHz, CD₃OD) δ 7.47-7.45(m, 4H), 7.33-7.31 (m, 6H), 7.12 (d, J=8.4 Hz, 2H), 6.86 (d, J=8.4 Hz,2H), 5.53 (s, 2H), 4.24-4.21 (m, 2H), 4.07-3.86 (m, 6H), 3.74-3.53 (m,4H), 3.34-2.53 (m, 16H), 1.90-1.30 (m, 20H), 1.42 (s, 36H).

Preparation of Compound 34

A solution of compound 33 (127 mg, 0.0834 mmol) and methyl3,5-diamino-6-chloropyrazine-2-carbonylcarbamimidothioate hydroiodicacid salt (7, 59 mg, 0.150 mmol) in EtOH (5.0 mL) was charged with DIPEA(108 mg, 0.839 mmol) at room temperature. The reaction mixture washeated at 70° C. for 2 h, cooled to room temperature, and concentratedin vacuum. The residue was purified by column chromatography (silicagel, 10:1 CH₂Cl₂/MeOH, 8:2:0.2 CHCl₃/MeOH/NH₄OH) to afford compound 34(81 mg, 65%) as a yellow solid: ¹H NMR (300 MHz, CD₃OD) δ 7.43-7.40 (m,4H), 7.31-7.30 (m, 6H), 7.10 (d, J=8.7 Hz, 2H), 6.83 (d, J=8.7 Hz, 2H),5.47 (s, 2H), 4.23-4.20 (m, 2H), 3.99-3.87 (m, 8H), 3.68-3.53 (m, 4H),3.34-3.15 (m, 4H), 3.05-2.95 (m, 10H), 2.81-2.51 (m, 10H), 1.66-1.32 (m,20H), 1.42 (s, 36H).

Preparation the Hydrochloride Salt of(S,R,R,R,2S,2′S)—N,N′-(3,3′-(2-(4-(4-(3-(3,5-diamino-6-chloropyrazine-2-carbonyl)guanidino)butyl)phenoxy)ethylazanediyl)bis(propane-3,1-diyl))bis(6-amino-2-((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexylamino)hexanamide)-(Compound35)

A solution of compound 34 (80.0 mg, 0.0535 mmol) in EtOH (1.0 mL) wascharged with 4 N aqueous HCl (3.0 mL) at room temperature and thereaction mixture was stirred for 4 h at room temperature. The reactionmixture was concentrated in vacuum and the residue was purified byreverse-phase column chromatography and lyophilized to affordhydrochloric acid salt 35 (39.0 mg, 55%) as a yellow hygroscopic solid:¹H NMR (400 MHz, D₂O) δ 7.23 (d, J=8.4 Hz, 2H), 6.92 (d, J=8.4 Hz, 2H),4.29 (br s, 2H), 4.08-4.03 (m, 2H), 3.90 (t, J=6.8 Hz, 2H), 3.76-3.59(m, 12H), 3.38-3.18 (m, 12H), 3.10-2.93 (m, 6H), 2.60 (br s, 2H),2.10-1.91 (m, 8H), 1.67-1.64 (m, 8H), 1.40-1.36 (m, 4H). HRMS calculatedfor C₄₈H₈₈ClN₁₄O₁₄ [M+H]⁺, 1119.6287. found 1119.6316. Elementalanalysis: % calculated C, 43.07; H, 7.00; N, 14.65. found C, 38.78; H,7.09; N, 13.03.

Preparation of Compound 36

A solution of compound 25 (2.19 g, 1.61 mmol) in EtOH (50 mL) wascharged with 4 N HCl in dioxane (10 mL) and the reaction mixture wasstirred for 3 h at room temperature. The reaction mixture wasconcentrated to afford hydrochloric acid salt 36 (1.88 g, 92%) as ayellow oil: ¹H NMR (300 MHz, CD₃OD) δ 7.78-7.75 (m, 4H), 7.62-7.60 (m,4H), 7.39-7.25 (m, 13H), 6.96 (d, J=8.1 Hz, 2H), 6.82 (d, J=8.1 Hz, 2H),5.04 (s, 2H), 4.37-4.15 (m, 12H), 3.73-3.06 (m, 8H), 2.99-2.81 (m, 6H),2.50-2.46 (m, 2H), 1.94-1.17 (m, 20H).

Preparation of Compound 37

A solution of compound 36 (1.86 g, 1.46 mmol) in EtOH (80 mL) wascharged with Goodmann's reagent (1.26 g, 3.23 mmol) and TEA (1.18 g,11.6 mmol). The reaction mixture was stirred at 0° C. for 2 h and atroom temperature for 3 h. After the solvent was removed, the residue waspurified by column chromatography (silica gel, 20:1 CH₂Cl₂/MeOH) toafford compound 37 (1.54 g, 64%) as a white semisolid: ¹H NMR (300 MHz,CD₃OD) δ 7.78-7.75 (m, 4H), 7.59-7.57 (m, 4H), 7.37-7.23 (m, 13H), 6.97(d, J=8.1 Hz, 2H), 6.73 (d, J=8.1 Hz, 2H), 5.04 (s, 2H), 4.36-3.95 (m,12H), 3.30-3.06 (m, 10H), 2.58-2.46 (m, 6H), 1.66-1.28 (m, 20H), 1.43(s, 36H).

Preparation of Compound 38

A solution of compound 37 (1.63 g, 0.99 mmol) in EtOH (24 mL) wascharged with piperidine (8.0 mL). The reaction mixture was stirred atroom temperature for 16 h. After the solvent was removed, the residuewas precipitated from MTBE/hexanes to afford compound 38 (1.01 g, 85%)as an off-white solid: ¹H NMR (300 MHz, CD₃OD) δ 7.33-7.32 (m, 5H), 7.06(d, J=8.4 Hz, 2H), 6.82 (d, J=8.4 Hz, 2H), 5.05 (s, 2H), 4.02 (br s,2H), 3.59-2.99 (m, 12H), 2.85 (br s, 2H), 2.62-2.54 (m, 6H), 1.71-1.28(m, 20H), 1.43 (s, 36H).

Preparation of Compound 39

A solution of compound 38 (120 mg, 0.100 mmol) in MeOH (5.0 mL) wascharged with compound 31 (67 mg, 0.250 mmol), AcOH (30 mg, 0.500 mmol),and NaCNBH₃ (29 mg, 0.400 mmol). After the reaction mixture was stirredat room temperature for 16 h, the solvent was removed in vacuum. Theresidue was purified by column chromatography (silica gel, 20:1CH₂Cl₂/MeOH, 10:1:0.1 CHCl₃/MeOH/NH₄OH) to afford compound 39 (101 mg,61%) as a white semisolid: ¹H NMR (300 MHz, CD₃OD) δ 7.47-7.44 (m, 4H),7.32-7.30 (m, 11H), 7.06 (d, J=8.4 Hz, 2H), 6.82 (d, J=8.4 Hz, 2H), 5.48(s, 2H), 5.05 (s, 2H), 4.23-4.20 (m, 2H), 4.01-3.85 (m, 8H), 3.71-3.58(m, 6H), 3.29-3.07 (m, 10H), 2.85-2.53 (m, 12H), 1.71-1.50 (m, 20H),1.47 (s, 18H), 1.47 (s, 18H).

Preparation of Compound 40

A suspension of compound 39 (518 mg, 0.304 mmol) and 10% Pd/C (250 mg)in EtOH (15 mL) and AcOH (3.0 mL) was subjected to hydrogenationconditions (1 atm) for 36 h at room temperature. The reaction mixturewas filtered through celite and washed with EtOH. The filtrate wasconcentrated, neutralized with 1 N Na₂CO₃, and purified by columnchromatography (silica gel, 10:1 CH₂Cl₂/MeOH, 5:1:0.1 CHCl₃/MeOH/NH₄OH)to afford compound 40 (283 mg, 54%) as a colorless oil: ¹H NMR (300 MHz,CD₃OD) δ 7.47-7.45 (m, 4H), 7.31-7.29 (m, 6H), 7.10 (d, J=8.4 Hz, 2H),6.85 (d, J=8.4 Hz, 2H), 5.49 (s, 2H), 4.25-4.20 (m, 2H), 4.03-3.89 (m,8H), 3.71-3.58 (m, 6H), 3.29-3.07 (m, 10H), 2.85-2.53 (m, 12H), 1.95 (s,12H), 1.64-1.19 (m, 56H).

Preparation of Compound 41

A solution of compound 40 (283 mg, 0.156 mmol) and methyl3,5-diamino-6-chloropyrazine-2-carbonylcarbamimidothioate hydroiodicacid salt (7, 98 mg, 0.250 mmol) in t-BuOH (10 mL) was charged withDIPEA (161 mg, 1.25 mmol) at room temperature. The reaction mixture washeated at 70° C. for 2 h, cooled to room temperature, and concentratedin vacuum. The residue was purified by column chromatography (silicagel, 10:1 CH₂Cl₂/MeOH, 8:1:0.1 CHCl₃/MeOH/NH₄OH) to afford compound 41(130 mg, 41%) as a yellow solid: ¹H NMR (400 MHz, CD₃OD) δ 7.45-7.44 (m,4H), 7.32-7.30 (m, 6H), 7.09 (d, J=8.4 Hz, 2H), 6.83 (d, J=8.4 Hz, 2H),5.47 (s, 2H), 4.23-4.20 (m, 2H), 4.00-3.85 (m, 8H), 3.68-3.58 (m, 6H),3.29-3.07 (m, 10H), 2.81-2.53 (m, 12H), 1.69-1.13 (m, 20H), 1.50 (s,18H), 1.45 (s, 18H).

Preparation of the Hydrochloride Salt of((S,R,R,R,2S,2′S)—N,N′-(3,3′-(2-(4-(4-(3-(3,5-diamino-6-chloropyrazine-2-carbonyl)guanidino)butyl)phenoxy)ethylazanediyl)bis(propane-3,1-diyl))bis(6-guanidino-2-((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexylamino)hexanamide)-Compound42

A solution of compound 41 (152 mg, 0.0853 mmol) in CH₂Cl₂ (6.0 mL) wascharged with TFA (2.0 mL) and the reaction mixture was stirred for 2 hat room temperature. After the solvent was removed, 4 N HCl (5.0 mL) wascharged to the residue and the reaction mixture was stirred for 4 h atroom temperature. After the solvent was removed, the residue waspurified by preparative HPLC and lyophilized to afford hydrochloric acidsalt 42 (39 mg, 38%) as a yellow hygroscopic solid: ¹H NMR (400 MHz,D₂O) δ 7.21 (d, J=8.4 Hz, 2H), 6.91 (d, J=8.4 Hz, 2H), 4.28 (br s, 2H),4.08-4.04 (m, 2H), 3.90 (t, J=6.8 Hz, 2H), 3.77-3.59 (m, 12H), 3.30-3.06(m, 18H), 2.59 (br s, 2H), 2.03-2.01 (m, 4H), 1.87-1.85 (m, 4H), 1.66(br s, 4H), 1.54-1.51 (m, 4H), 1.35-1.31 (m, 4H). HRMS calculated forC₅₀H₉₂ClN₁₈O₁₄ [M+H]⁺, 1203.6723. found 1203.6818.

Preparation of Compound 43

A suspension of compound 22 (7.00 g, 10.7 mmol) and 10% Pd/C (3.00 g) inEtOH/AcOH (70 mL/2.0 mL) was subjected to hydrogenation conditions (1atm) for 6 h at room temperature. The reaction mixture was filteredthrough celite and washed with EtOH. The filtrate was concentrated invacuum to afford acetic salt 43 (7.00 g, crude) as an off-white solid.The crude product was used directly in the next step. ¹H NMR (400 MHz,CDCl₃) 7.13 (d, J=8.4 Hz, 2H), 6.81 (d, J=8.4 Hz, 2H), 4.09 (t, J=5.2Hz, 2H), 3.16-3.10 (m, 4H), 3.01 (t, J=5.2 Hz, 2H), 2.89 (t, J=6.8 Hz,2H), 2.73 (t, J=6.8 Hz, 4H), 2.56 (t, J=6.8 Hz, 2H), 1.75-1.63 (m, 8H),1.67-1.65 (m, 6H), 1.42 (s, 18H).

Preparation of Compound 44

A solution of amine salt 43 (7.00 g, crude) and methyl3,5-diamino-6-chloropyrazine-2-carbonylcarbamimidothioate hydroiodicacid salt (7, 5.41 g, 14.4 mmol) in EtOH (70 mL) was charged with DIPEA(14.0 g, 108 mmol) at room temperature. The reaction mixture was heatedat 70° C. in a sealed tube for 2 h, cooled to room temperature, andconcentrated in vacuum. The residue was purified by columnchromatography (silica gel, 80:18:2 CHCl₃/CH₃OH/NH₄OH) to affordguanidine 44 (3.00 g, 38% over 2 steps) as a yellow solid: ¹H NMR (400MHz, CD₃OD) 7.10 (d, J=8.4 Hz, 2H), 6.85 (d, J=8.4 Hz, 2H), 4.02 (t,J=5.6 Hz, 2H), 3.08 (t, J=6.8 Hz, 4H), 2.83 (t, J=5.6 Hz, 2H), 2.61-2.55(m, 6H), 1.68-1.63 (m, 8H), 1.40 (s, 18H).

Preparation of the Hydrochloride Salt of3,5-diamino-N—(N-(4-(4-(2-(bis(3-aminopropyl)amino)ethoxy)phenyl)butyl)carbamimidoyl)-6-chloropyrazine-2-carboxamide-Compound45

4 N HCl in water (20.0 mL) and ethanol (10.0 mL) was charged withcompound 44 (1.80 g, 2.45 mmol) and the reaction mixture was stirred atroom temperature for 5 h. The solvent was removed and the mixture waspurified by reverse-phase column to give compound 45 (1.30 g, 78%) as ayellow solid: ¹H NMR (400 MHz, D₂O) 7.20 (d, J=8.8 Hz, 2H), 6.91 (d,J=8.8 Hz, 2H), 4.30 (t, J=4.4 Hz, 2H), 3.66 (t, J=4.4 Hz, 2H), 3.37 (t,J=8.0 Hz, 4H), 3.25 (t, J=6.4 Hz, 2H), 3.05 (t, J=8.0 Hz, 4H), 2.57 (d,J=6.4 Hz, 2H), 2.19-2.11 (m, 4H), 1.63 (br s, 4H).

Preparation of3,5-diamino-N—(N-(4-(4-((R)-11-amino-17-(3-((R)-2-amino-6-guanidinohexanamido)propyl)-5-imino-3,12-dioxo-2-oxa-4,6,13,17-tetraazanonadecan-19-yloxy)phenyl)butyl)carbamimidoyl)-6-chloropyrazine-2-carboxamide-Compound46 was isolated as a byproduct of the preparation of Compound 9. ¹H NMR(300 MHz, D₂O) δ 7.26 (d, J=8.4 Hz, 2H), 6.91 (d, J=8.4 Hz, 2H), 4.40(br s, 2H), 3.88-3.85 (m, 2H), 3.79 (br s, 2H), 3.34-3.33 (m, 10H), 3.15(s, 3H), 3.12 (t, J=6.6 Hz, 4H), 2.63 (br s, 2H), 2.06-2.04 (m, 4H),1.83-1.78 (m, 4H), 1.69 (br s, 4H), 1.57-1.50 (m, 4H), 1.38-1.33 (m,4H).

Preparation of Compound 47

A solution of amino acid 11 (750 mg, 1.53 mmol) in CH₂Cl₂ (30 mL) wascharged with EEDQ (890 mg, 2.97 mmol), bis-amine 4 (1.73 g, 3.06 mmol)and NMM (2.40 g, 23.7 mmol). The reaction mixture was stirred at 0° C.for 6 h and at room temperature for 24 h. Additional amino acid 11 (750mg, 1.53 mmol) and EEDQ (890 mg, 2.97 mmol) were added and the resultingmixture was stirred at room temperature for 24 h. The solvent wasremoved and the residue was purified by column chromatography (silicagel, 10:1 CH₂Cl₂/MeOH) to afford compound 47 (620 mg, mixture withcompound 11) as a yellow solid, which was used directly in the nextstep.

Preparation of Compound 48

A solution of compound 47 (850 mg, mixture with compound 11) in THF (6.0mL), MeOH (6.0 mL), and water (2.0 mL) was charged with NaHCO₃ (462 mg,5.52 mmol) and Boc₂O (250 mg, 1.14 mmol). The reaction mixture wasstirred for 6 h at room temperature. After the solvent was removed, theresidue was partitioned between CH₂Cl₂(20 mL) and water (20 mL). Theaqueous layer was separated and extracted with CH₂Cl₂ (20 mL). Thecombined organic extracts were dried over Na₂SO₄, concentrated, andpurified by column chromatography (silica gel, 10:1 CH₂Cl₂/MeOH) toafford compound 48 (460 mg, 11% over 2 steps) as a white solid: ¹H NMR(300 MHz, CD₃OD) δ 7.33-7.32 (m, 5H), 7.06 (d, J=8.4 Hz, 2H), 6.83 (d,J=8.4 Hz, 2H), 5.05 (s, 2H), 4.06 (t, J=5.7 Hz, 2H), 3.95 (br s, 1H),3.34-3.23 (m, 4H), 3.14-3.07 (m, 4H), 2.92 (br s, 2H), 2.66-2.52 (m,6H), 1.86-1.54 (m, 14H), 1.51 (s, 9H), 1.46 (s, 9H), 1.42 (s, 9H), 1.40(s, 9H).

Preparation of Compound 49

A suspension of compound 48 (460 mg, 0.448 mmol) and 10% Pd/C (230 mg)in EtOH (15 mL) was subjected to hydrogenation conditions (1 atm) for 3h at room temperature. The reaction mixture was filtered through celiteand washed with EtOH. The filtrate was concentrated and purified bycolumn chromatography (silica gel, 10:1 CH₂Cl₂/MeOH) to afford compound49 (342 mg, 86%) as a white solid: ¹H NMR (300 MHz, CD₃OD) δ 7.09 (d,J=8.1 Hz, 2H), 6.85 (d, J=8.1 Hz, 2H), 4.03 (t, J=5.4 Hz, 2H), 3.95 (brs, 1H), 3.34-3.24 (m, 4H), 3.08 (d, J=6.6 Hz, 2H), 2.88-2.84 (m, 4H),2.59-2.52 (m, 6H), 1.64-1.57 (m, 14H), 1.52 (s, 9H), 1.46 (s, 9H), 1.43(s, 9H), 1.41 (s, 9H).

Preparation of Compound 50

A solution of amine 49 (342 mg, 0.383 mmol) and methyl3,5-diamino-6-chloropyrazine-2-carbonylcarbamimidothioate hydroiodicacid salt (7, 238 mg, 0.611 mmol) in t-BuOH (15 mL) was charged withDPEA (392 mg, 3.04 mmol) at room temperature. The reaction mixture washeated at 70° C. for 2 h, cooled to room temperature, and concentratedin vacuum. The residue was purified by column chromatography (silicagel, 10:1 CH₂Cl₂/MeOH, 5:1:0.1 CHCl₃/MeOH/NH₄OH) to afford compound 50(236 mg, 56%) as a yellow solid: ¹H NMR (300 MHz, CD₃OD) δ 7.09 (d,J=7.2 Hz, 2H), 6.85 (d, J=7.2 Hz, 2H), 4.03 (d, J=5.1 Hz, 2H), 3.95 (brs, 1H), 3.31-3.25 (m, 6H), 3.08 (t, J=6.3 Hz, 2H), 2.84 (t, J=4.8 Hz,2H), 2.60-2.56 (m, 6H), 1.67-1.54 (m, 14H), 1.51 (s, 9H), 1.46 (s, 9H),1.42 (s, 9H), 1.40 (s, 9H).

Preparation of the Hydrochloride Acid Salt of(S)-3,5-diamino-N—(N-(4-(4-(2-((3-(2-amino-6-guanidinohexanamido)propyl)(3-aminopropyl)amino)ethoxy)phenyl)butyl)carbamimidoyl)-6-chloropyrazine-2-carboxamide-Compound51

A solution of compound 50 (235 mg, 0.212 mmol) in EtOH (1.5 mL) wascharged with 4 N aqueous HCl (5.0 mL) at room temperature, and thereaction mixture was stirred for 4 h at room temperature. The reactionmixture was concentrated in vacuum and the residue was purified byreverse-phase column chromatography and lyophilized to affordhydrochloric acid salt 51 (145 mg, 76%) as a yellow hygroscopic solid:¹H NMR (400 MHz, D₂O) δ 7.22 (d, J=8.4 Hz, 2H), 6.91 (d, J=8.4 Hz, 2H),4.29 (t, J=4.8 Hz, 2H), 3.88 (t, J=6.8 Hz, 2H), 3.63 (t, J=4.4 Hz, 2H),3.35-3.28 (m, 8H), 3.09-3.03 (m, 2H), 2.59 (br s, 2H), 2.17-2.12 (m,2H), 2.03-1.97 (m, 2H), 1.83-1.79 (m, 2H), 1.66 (br s, 4H), 1.53-1.49(m, 2H), 1.34-1.32 (m, 2H). HRMS calculated for C₃₁H₅₄ClN₁₄O₃[M+H]⁺,705.4186. found 705.4216.

Preparation of the Hydrochloride Salt of(S,2S,2′S)—N,N′-(3,3′-(2-(4-(4-(3-(3,5-diamino-6-chloropyrazine-2-carbonyl)guanidino)butyl)phenoxy)ethylazanediyl)bis(propane-3,1-diyl))bis(2-amino-6-((S)-2,6-diaminohexanamido)hexanamide)

Preparation of Compound 54

A solution of amino acid 52 (2.00 g, 5.77 mmol) in CH₂Cl₂ (40 mL) wascharged with EEDQ (2.07 g, 6.92 mmol), compound 53 (1.71 g, 5.77 mmol),and NMM (1.74 g, 17.3 mmol). The reaction mixture was stirred at roomtemperature for 16 h. The solvent was removed and the residue waspurified by column chromatography (silica gel, 10:1 CH₂Cl₂/EtOAc, 10:1CH₂Cl₂/MeOH) to afford compound 54 (2.80 g, 82%) as a colorless oil: ¹HNMR (300 MHz, CD₃OD) δ 4.08-4.04 (m, 1H), 3.96-3.94 (m, 1H), 3.70 (s,3H), 3.21-3.15 (m, 2H), 3.02 (t, J=6.6 Hz, 2H), 1.73-1.47 (m, 12H), 1.46(s, 27H).

Preparation of Compound 55

A solution of compound 54 (24.8 g, 42.0 mmol) in THF (200 mL), MeOH (200mL), and H₂O (60 mL) was charged with NaOH (16.8 g, 420 mmol). Thereaction mixture was stirred at room temperature for 3 h. The solventwas removed and water (300 mL) was charged to the residue. After the pHwas adjusted to 5 with 1 N HCl, the resulting solid was filtered out anddried to afford compound 55 (22.5 g, 93%) as an orange solid: ¹H NMR(400 MHz, CD₃OD) δ4.06-4.02 (m, 1H), 3.96-3.94 (m, 1H), 3.70 (s, 3H),3.21-3.18 (m, 1H), 3.02 (t, J=6.8 Hz, 2H), 1.80-1.53 (m, 12H), 1.43 (s,27H).

Preparation of Compound 56

A solution of amino acid 55 (2.54 g, 4.41 mmol) in CH₂Cl₂ (80 mL) wascharged with EEDQ (1.58 g, 5.28 mmol), compound 4 (1.25 g, 2.20 mmol),and NMM (3.55 g, 35.2 mmol). The reaction mixture was stirred at roomtemperature for 16 h. The solvent was removed and the residue waspurified by column chromatography (silica gel, 10:1 CH₂Cl₂/EtOAc, 10:1CH₂Cl₂/MeOH) to afford compound 56 (2.40 g, 75%) as a colorless oil: ¹HNMR (300 MHz, CD₃OD) δ 7.32-7.28 (m, 5H), 7.06 (d, J=8.4 Hz, 2H), 6.82(d, J=8.4 Hz, 2H), 5.05 (s, 2H), 4.04-3.95 (m, 6H), 3.24-2.99 (m, 14H),2.87 (br s, 2H), 2.63-2.52 (m, 6H), 1.72-1.42 (m, 32H), 1.47 (s, 54H).

Preparation of Compound 57

A suspension of compound 56 (2.40 g, 1.52 mmol) and 10% Pd/C (1.20 g) inEtOH (50 mL) and AcOH (2.0 mL) was subjected to hydrogenation conditions(1 atm) for 6 h at room temperature. The reaction mixture was filteredthrough celite and washed with EtOH. The filtrate was concentrated andwashed with MTBE to afford compound 57 (2.13 g, 90%) as a colorless oil:¹H NMR (300 MHz, CD₃OD) δ 7.12 (d, J=8.4 Hz, 2H), 6.90 (d, J=8.4 Hz,2H), 4.19 (br s, 2H), 3.94-3.85 (m, 4H), 3.34-2.92 (m, 20H), 2.62 (t,J=6.6 Hz, 2H), 1.95 (s, 6H), 1.90-1.50 (m, 32H), 1.47 (s, 54H).

Preparation of Compound 58

A solution of amine 37 (2.12 g, 1.36 mmol) and methyl3,5-diamino-6-chloropyrazine-2-carbonylcarbamimidothioate hydroiodicacid salt (7, 638 mg, 1.63 mmol) in EtOH (80 mL) was charged with DIPEA(1.41 g, 10.8 mmol) at room temperature. The reaction mixture was heatedat 70° C. for 2 h, cooled to room temperature, and concentrated invacuum. The residue was purified by column chromatography (silica gel,10:1 CH₂Cl₂/MeOH, 5:1:0.1 CHCl₃/MeOH/NH₄OH) to afford compound %8 (1.21g, 54%) as a yellow solid: ¹H NMR (400 MHz, CD₃OD) δ7.10 (d, J=8.4 Hz,2H), 6.85 (d, J=8.4 Hz, 2H), 4.13 (t, J=5.2 Hz, 2H), 3.94-3.85 (m, 4H),3.34-3.10 (m, 10H), 3.01 (t, J=6.8 Hz, 4H), 2.89 (br s, 2H), 2.62 (t,J=6.4 Hz, 6H), 1.92-1.47 (m, 32H), 1.43 (s, 54H).

Preparation of Compound 59—The Hydrochloride Salt of(S,2S,2′S)—N,N′-(3,3′-(2-(4-(4-(3-(3,5-diamino-6-chloropyrazine-2-carbonyl)guanidino)butyl)phenoxy)ethylazanediyl)bis(propane-3,1-diyl))bis(2-amino-6-((S)-2,6-diaminohexanamido)hexanamide)

A solution of compound 58 (360 mg, 0.218 mmol) in EtOH (2.0 mL) wascharged with 4 N aqueous HCl (6.0 mL) at room temperature and thereaction mixture was stirred for 6 h at room temperature. The reactionmixture was concentrated in vacuum and the residue was purified byreverse-phase column chromatography and lyophilized to affordhydrochloric acid salt 59 (117 mg, 41%) as a yellow hygroscopic solid:¹H NMR (400 MHz, D₂O) δ 7.22 (d, J=8.4 Hz, 2H), 6.91 (d, J=8.4 Hz, 2H),4.28 (br s, 2H), 3.90-3.86 (m, 4H), 3.60 (br s, 2H), 3.37-3.15 (m, 14H),2.60 (br s, 2H), 2.00-1.96 (m, 4H), 1.85-1.79 (m, 8H), 1.68-1.64 (m,8H), 1.49-1.32 (m, 12H). HRMS calculated for C₄₈H₈₈ClN₁₈O₆ [M+H]+,1047.6817. found 1047.6831.

Preparation of The Hydrochloride Salt of(S,S,2S,2′S)—N,N′-(3,3′-(2-(4-(4-(3-(3,5-diamino-6-chloropyrazine-2-carbonyl)guanidino)butyl)phenoxy)ethylazanediyl)bis(propane-3,1-diyl))bis(2-amino-6-((S)-2-amino-6-((S)-2,6-diaminohexanamido)hexanamido)hexanamide)Scheme 10

Preparation of Compound 60

A solution of amino acid 55 (12.0 g, 20.8 mmol) in CH₂Cl₂ (300 mL) wascharged with EEDQ (7.40 g, 25.0 mmol), compound 33 (6.20 g, 20.8 mmol),and NMM (6.30 g, 62.4 mmol). The reaction mixture was stirred at roomtemperature for 16 h. The solvent was removed and the residue waspurified by column chromatography (silica gel, 10:1 CH₂Cl₂/EtOAc, 10:1CH₂Cl₂/MeOH) to afford compound 40 (13.1 g, 77%) as a yellow oil: ¹H NMR(300 MHz, CD₃OD) δ 4.08 (br s, 1H), 3.94 (br s, 2H), 3.70 (s, 3H),3.21-3.15 (m, 4H), 3.02 (t, J=6.6 Hz, 2H), 1.70-1.47 (m, 18H), 1.43 (s,36H).

Preparation of Compound 61

A solution of compound 60 (13.0 g, 15.9 mmol) in THF (100 mL), MeOH (100mL), and H₂O (35 mL) was charged with NaOH (3.20 g, 80.0 mmol). Thereaction mixture was stirred at room temperature for 4 h. The solventwas removed and water (300 mL) was charged to the residue. After the pHwas adjusted to 5 with 1 N HCl, the resulting solid was filtered out anddried to afford compound 61 (12.1 g, 95%) as an orange solid: ¹H NMR(300 MHz, CD₃OD) δ 4.03 (br s, 1H), 3.94 (br s, 2H), 3.70 (s, 3H),3.29-3.14 (m, 4H), 3.02 (t, J=6.6 Hz, 2H), 1.70-1.47 (m, 18H), 1.43 (s,36H).

Preparation of Compound 62

A solution of amino acid 61 (500 mg, 0.622 mmol) in CH₂Cl₂ (15 mL) wascharged with EEDQ (223 mg, 0.746 mmol), compound 4 (176 mg, 0.311 mmol),and NMM (502 mg, 4.97 mmol). The reaction mixture was stirred at roomtemperature for 16 h. The solvent was removed and the residue waspurified by column chromatography (silica gel, 10:1 CH₂Cl₂/EtOAc, 10:1CH₂Cl₂/MeOH) to afford compound 62 (290 mg, 46%) as a colorless oil: ¹HNMR (400 MHz, CD₃OD) δ 7.33-7.29 (m, 5H), 7.12 (d, J=8.4 Hz, 2H), 6.91(d, J=8.4 Hz, 2H), 5.05 (s, 2H), 4.32 (br s, 2H), 3.94-3.84 (m, 6H),3.57 (br s, 2H), 3.32-3.00 (m, 22H), 2.57 (t, J=7.2 Hz, 2H), 1.70-1.47(m, 44H), 1.47 (s, 72H).

Preparation of Compound 63

A suspension of compound 62 (2.20 g, 1.08 mmol) and 10% Pd/C (1.10 g) inEtOH (50 mL) was subjected to hydrogenation conditions (1 atm) for 6 hat room temperature. The reaction mixture was filtered through celiteand washed with EtOH. The filtrate was concentrated to afford compound63 (1.87 g, 91%) as a white solid: ¹H NMR (300 MHz, CD₃OD) δ 7.16 (d,J=8.4 Hz, 2H), 6.93 (d, J=8.4 Hz, 2H), 4.30 (br s, 2H), 3.91-3.83 (m,6H), 3.52 (br s, 2H), 3.17-2.94 (m, 22H), 2.62 (s, 2H), 1.98-1.47 (m,44H), 1.47 (s, 72H).

Preparation of Compound 64

A solution of amine 43 (1.86 g, 0.980 mmol) and methyl3,5-diamino-6-chloropyrazine-2-carbonylcarbamimidothioate hydroiodicacid salt (7, 455 mg, 1.17 mmol) in EtOH (20 mL) was charged with DIPEA(1.01 g, 7.78 mmol) at room temperature. The reaction mixture was heatedat 70° C. for 2 h, cooled to room temperature, and concentrated invacuum. The residue was purified by column chromatography (silica gel,10:1 CH₂Cl₂/MeOH, 5:1:0.1 CHCl₃/MeOH/NH₄OH) to afford compound 64 (1.26g, 61%) as a yellow solid: ¹H NMR (300 MHz, CD₃OD) δ 7.11 (d, J=8.7 Hz,2H), 6.84 (d, J=8.7 Hz, 2H), 4.05-3.95 (br s, 8H), 3.34-3.07 (m, 14H),3.02 (t, J=6.6 Hz, 4H), 2.85 (t, J=5.4 Hz, 2H), 2.62-2.57 (m, 6H),1.69-1.47 (m, 44H), 1.43 (s, 72H).

Preparation of The Hydrochloride Salt of(S,S,2S,2′S)—N,N′-(3,3′-(2-(4-(4-(3-(3,5-diamino-6-chloropyrazine-2-carbonyl)guanidino)butyl)phenoxy)ethylazanediyl)bis(propane-3,1-diyl))bis(2-amino-6-((S)-2-amino-6-((S)-2,6-diaminohexanamido)hexanamido)hexanamide)-Compound65

A solution of compound 64 (1.25 g, 0.661 mmol) in EtOH (5.0 mL) wascharged with 4 N aqueous HCl (15 mL) at room temperature and thereaction mixture was stirred for 4 h at room temperature. The reactionmixture was concentrated in vacuum, and the residue was purified byreverse-phase column chromatography and lyophilized to affordhydrochloric acid salt 65 (681 mg, 62%) as a yellow hygroscopic solid:¹H NMR (400 MHz, D₂O) δ 7.22 (d, J=8.4 Hz, 2H), 6.91 (d, J=8.4 Hz, 2H),4.28 (br s, 2H), 3.91-3.87 (m, 6H), 3.60 (br s, 2H), 3.37-3.17 (m, 18H),2.97-2.93 (m, 4H), 2.59 (br s, 2H), 2.01-1.96 (m, 4H), 1.84-1.79 (m,12H), 1.68-1.64 (m, 8H), 1.52-1.32 (m, 20H). HRMS calculated forC₆₀H₁₁₂ClN₂₂O₈ [M+H]⁺, 1303.8717. found 1303.8708.

10. Preparation of the Hydrochloride Salt ofN,N′-((7S,19S)-7,19-diamino-13-(2-(4-(4-(3-(3,5-diamino-6-chloropyrazine-2-carbonyl)guanidino)butyl)phenoxy)ethyl)-1,25-diimino-8,18-dioxo-2,9,13,17,24-pentaazapentacosane-1,25-diyl)dibenzamide

Preparation of Compound 68

A solution of compound 66 (300 mg, 2.05 mmol) and DIPEA (2.10 g, 16.4mmol) in CH₂Cl₂ (6.0 mL) was charged with compound 67 (316 mg, 2.25mmol). The reaction mixture was stirred at room temperature for 24 h.Water (20 mL) was added, and the aqueous layer was extracted with CH₂Cl₂(20 mL). The combined organic extracts were dried over Na₂SO₄,concentrated, and purified by column chromatography (silica gel, 10:1CH₂Cl₂/MeOH) to afford compound 68 (340 mg, 78%) as a white solid: ¹HNMR (300 MHz, CDCl₃) δ 10.0 (br s, 1H), 8.64 (d, J=2.7 Hz, 1H), 8.30(dd, J=0.9, 8.1 Hz, 2H), 7.74 (d, J=0.9 Hz, 1H), 7.53-7.45 (m, 3H), 6.48(dd, J=1.8, 2.7 Hz, 1H).

Preparation of Compound 70

A solution of compound 69 (200 mg, 0.813 mmol) in MeOH (8.0 mL) wascharged with compound 68 (174 mg, 0.813 mmol) and DIPEA (419 mg, 3.25mmol). The reaction mixture was stirred at room temperature for 16 h.Additional 68 (35 mg, 0.162 mmol) was charged and the reaction mixturewas stirred at room temperature for 5 h. After the solvent was removed,the residue was purified by column chromatography (silica gel, 10:1CH₂Cl₂/MeOH) to afford compound 70 (246 mg, 78%) as a white solid: ¹HNMR (300 MHz, CD₃OD) δ 8.00-7.98 (m, 2H), 7.65-7.53 (m, 3H), 4.03 (br s,1H), 3.34-3.29 (m, 2H), 1.85-1.49 (m, 6H), 1.42 (s, 9H).

Preparation of Compound 72

A solution of amino acid 70 (200 mg, 0.509 mmol) in CH₂C₂(5.0 mL) wascharged with EEDQ (305 mg, 1.02 mmol) and compound 71 (116 mg, 0.255mmol). The reaction mixture was stirred at room temperature for 30 h.Additional 70 (40 mg, 0.118 mmol) was charged and the reaction mixturewas stirred at room temperature for 6 h. The solvent was removed and theresidue was purified by column chromatography (silica gel, 10:1CH₂Cl₂/MeOH) to afford compound 72 (197 mg, 64%) as a colorless oil: ¹HNMR (400 MHz, CD₃OD) δ 8.04 (br s, 4H), 7.45-7.28 (m, 11H), 7.05 (d,J=8.4 Hz, 2H), 6.82 (d, J=8.4 Hz, 2H), 5.05 (s, 2H), 4.04-3.98 (m, 4H),3.24-3.23 (m, 8H), 3.09 (t, J=6.8 Hz, 2H), 2.92 (br s, 2H), 2.65 (br s,4H), 2.52 (t, J=7.6 Hz, 2H), 1.72-1.46 (m, 20H), 1.41 (s, 18H).

Preparation of Compound 73

A suspension of compound 72 (195 mg, 0.162 mmol) and 10% Pd/C (100 mg)in EtOH (5.0 mL) was subjected to hydrogenation conditions (1 atm) for 4h at room temperature. The reaction mixture was filtered through celiteand precipitated from MTBE/hexanes. The filtrate was concentrated toafford compound 73 (149 mg, 86%) as a white solid: ¹H NMR (400 MHz,CD₃OD) δ 8.05 (br s, 4H), 7.45-7.34 (m, 6H), 7.08 (d, J=8.4 Hz, 2H),6.82 (d, J=8.4 Hz, 2H), 4.00-3.98 (m, 4H), 3.34-3.23 (m, 8H), 2.81-2.79(m, 4H), 2.55 (br s, 6H), 1.64-1.42 (m, 20H), 1.41 (s, 18H).

Preparation of Compound 74

A solution of amine 73 (145 mg, 0.135 mmol) and methyl3,5-diamino-6-chloropyrazine-2-carbonylcarbamimidothioate hydroiodicacid salt (7, 64 mg, 0.162 mmol) in EtOH (3.0 mL) was charged with DIPEA(88 mg, 0.675 mmol) at room temperature. The reaction mixture was heatedat 70° C. for 2 h, cooled to room temperature, and concentrated invacuum. The residue was purified by column chromatography (silica gel,10:1 CH₂Cl₂/MeOH, 5:1:0.1 CHCl₃/MeOH/NH₄OH) to afford compound 74 (104mg, 60%) as a yellow solid: ¹H NMR (400 MHz, CD₃OD) δ 8.05 (br s, 4H),7.42-7.33 (m, 6H), 7.08 (d, J=8.4 Hz, 2H), 6.82 (d, J=8.4 Hz, 2H),4.00-3.98 (m, 4H), 3.34-3.23 (m, 10H), 2.80-2.79 (m, 2H), 2.58-2.53 (m,6H), 1.68-1.61 (m, 20H), 1.41 (s, 18H).

The preparation of the Hydrochloride salt ofN,N′-((7S,19S)-7,19-diamino-13-(2-(4-(4-(3-(3,5-diamino-6-chloropyrazine-2-carbonyl)guanidino)butyl)phenoxy)ethyl)-1,25-diimino-8,18-dioxo-2,9,13,17,24-pentaazapentacosane-1,25-diyl)dibenzamide-Compound75 A solution of compound 74 (1.02 g, 0.794 mmol) in EtOH (20 mL) wascharged with 4 N aqueous HCl (20 mL) at room temperature and thereaction mixture was stirred for 4 h at room temperature. The reactionmixture was concentrated in vacuum and the residue was purified byreverse-phase column chromatography and lyophilized to affordhydrochloric acid salt 75 (511 mg, 52%) as a yellow hygroscopic solid:¹H NMR (300 MHz, D₂O) δ 7.73 (d, J=7.5 Hz, 2H), 7.55 (t, J=7.5 Hz, 2H),7.55 (t, J=7.5 Hz, 4H), 7.13 (d, J=8.7 Hz, 2H), 6.82 (d, J=8.7 Hz, 2H),4.13 (br s, 2H), 3.85 (t, J=6.6 Hz, 2H), 3.47 (br s, 2H), 3.23-3.19 (m,14H), 2.49 (br s, 2H), 1.91-1.75 (m, 8H), 1.58-1.52 (m, 8H), 1.37-1.32(m, 4H). HRMS calculated for C₅₂H₇₆ClN₁₈O₆ [M+H]⁺, 1083.5878. found1083.5884.

Preparation of The Hydrochloride Salt of(2S,2′S,2″S,2′″S)—N,N′,N″,N′″-(3,3′,3″,3′″-(3,3′-(2-(4-(4-(3-(3,5-diamino-6-chloropyrazine-2-carbonyl)guanidino)butyl)phenoxy)ethylazanediyl)bis(propane-3,1-diyl))bis(azanetriyl)tetrakis(propane-3,1-diyl))tetrakis(2-amino-6-guanidinohexanamide)-Compound82

Preparation of Compound 77

A solution of compound 4 (100 mg, 0.219 mmol) in MeOH (4.0 mL) wascharged with compound 76 (227 mg, 1.37 mmol), NaCNBH₃ (128 mg, 1.76mmol), and AcOH (132 mg, 2.20 mmol). The reaction mixture was stirred atroom temperature for 16 h. Additional compound 76 (151 mg, 0.876 mmol),NaCNBH₃ (79.8 mg, 1.10 mmol), and AcOH (79 mg, 1.31 mmol) were added,and the resulting mixture was stirred at room temperature for 24 h. Thesolvent was removed and the residue was purified by columnchromatography (silica gel, 20:1 CH₂Cl₂/MeOH) to afford compound 77 (63mg, 27%) as a colorless oil: ¹H NMR (300 MHz, CD₃OD) δ 7.32-7.31 (m,5H), 7.12 (d, J=8.4 Hz, 2H), 6.86 (d, J=8.4 Hz, 2H), 5.05 (s, 2H), 4.10(d, J=4.5 Hz, 2H), 3.34-3.11 (m, 22H), 2.95 (br s, 2H), 2.78-2.75 (m,4H), 2.57 (d, J=7.8 Hz, 2H), 1.98-1.83 (m, 10H), 1.60-1.49 (m, 6H), 1.43(s, 36H).

Preparation of Compound 78

A solution of compound 77 (502 mg, 0.463 mmol) in EtOH (5.0 mL) wascharged with 4 N aqueous HCl (5.0 mL) at room temperature and thereaction mixture was stirred for 4 h at room temperature. The reactionmixture was concentrated in vacuum and the residue was precipitated fromMTBE to afford compound 78 (374 mg, 86%) as a colorless oil: ¹H NMR (400MHz, CD₃OD) δ 7.34-7.29 (m, 5H), 7.13 (d, J=7.6 Hz, 2H), 7.00 (d, J=7.6Hz, 2H), 5.06 (s, 2H), 4.45 (br s, 2H), 3.81-3.48 (m, 20H), 3.13-3.10(m, 10H), 2.59-2.43 (m, 5H), 2.26 (br s, 7H), 1.64-1.44 (m, 4H).

Preparation of Compound 79

A solution of amino acid 11 (104 mg, 0.213 mmol) in CH₂Cl₂ (5.0 mL) wascharged with EEDQ (127 mg, 0.426 mmol), compound 78 (50.0 mg, 0.0530mmol), and NMM (108 mg, 1.06 mmol). The reaction mixture was stirred atroom temperature for 4 days. The solvent was removed and the residue waspurified by column chromatography (silica gel, 10:1 CH₂Cl₂/MeOH, 5:1:0.1CHCl₃/MeOH/NH₄OH) to afford compound 79 (52 mg, 38%) as a colorless oil:¹H NMR (300 MHz, CD₃OD) δ 7.32-7.29 (m, 5H), 7.11 (d, J=7.8 Hz, 2H),6.86 (d. J=7.8 Hz, 2H), 5.06 (s, 2H), 4.14-3.96 (m, 6H), 3.30-2.80 (m,22H), 2.67-2.45 (m, 16H), 1.90-1.20 (m, 148H).

Preparation of Compound 80

A suspension of compound 79 (320 mg, 0.124 mmol) and 10% Pd/C (160 mg)in EtOH (10 mL) and AcOH (1.0 mL) was subjected to hydrogenationconditions (1 atm) for 16 h at room temperature. The reaction mixturewas filtered through celite and washed with EtOH. The filtrate wasconcentrated and precipitated from MTBE/hexanes to afford compound 80(285 mg, 87%) as a colorless oil: ¹H NMR (300 MHz, CD₃OD) δ 7.14 (d,J=8.4 Hz, 2H), 6.89 (d, J=8.4 Hz, 2H), 4.11-3.79 (m, 6H), 3.35-2.66 (m,38H), 1.90-1.20 (m, 148H).

Preparation of Compound 81

A solution of amine 80 (280 mg, 0.104 mmol) and methyl3,5-diamino-6-chloropyrazine-2-carbonylcarbamimidothioate hydroiodicacid salt (7, 49.0 mg, 0.125 mmol) in t-BuOH (10 mL) was charged withDIPEA (103 mg, 0.795 mmol) at room temperature. The reaction mixture washeated at 70° C. for 2 h, cooled to room temperature, and concentratedin vacuum. The residue was purified by column chromatography (silicagel, 10:1 CH₂Cl₂/MeOH, 5:1:0.1 CHCl₃/MeOH/NH₄OH) to afford compound 81(112 mg, 40%) as a yellow solid: ¹H NMR (300 MHz, CD₃OD) δ 7.11 (d,J=8.4 Hz, 2H), 6.83 (d, J=8.4 Hz, 2H), 4.03 (br s, 6H), 3.30-3.07 (m,20H), 2.88 (br s, 2H), 2.60-2.48 (m, 16H), 1.65-1.23 (m, 148H).

Preparation of The Hydrochloride Salt of(2S,2′S,2″S,2′″S)—N,N′,N″,N′″-(3,3′,3″,3′″-(3,3′-(2-(4-(4-(3-(3,5-diamino-6-chloropyrazine-2-carbonyl)guanidino)butyl)phenoxy)ethylazanediyl)bis(propane-3,1-diyl))bis(azanetriyl)tetrakis(propane-3,1-diyl))tetrakis(2-amino-6-guanidinohexanamide)-Compound82

A solution of compound 81 (15.0 mg, 0.00567 mmol) in EtOH (2.0 mL) wascharged with 4 N aqueous HCl (2.0 mL) at room temperature and thereaction mixture was stirred for 4 h at room temperature. The reactionmixture was concentrated in vacuum, and the residue was purified byreverse-phase column chromatography and lyophilized to affordhydrochloric acid salt 82 (3.95 mg, 37%) as a yellow hygroscopic solid:¹H NMR (400 MHz, D₂O) δ 7.24 (d, J=8.4 Hz, 2H), 6.96 (d, J=8.4 Hz, 2H),4.34 (br s, 2H), 3.93 (d, J=6.8 Hz, 2H), 3.64 (br s, 2H), 3.38-3.11 (m,34H), 2.60 (br s, 2H), 2.21-2.17 (m, 4H), 2.02-1.79 (m, 16H), 1.66-1.54(m, 12H), 1.41-1.28 (m, 8H). HRMS calculated for C₆₄H₁₂₄ClN₃₀O₆[M+H]⁺,1444.0003. found 1444.0054.

Preparation of The Hydrochloride Salt of3,5-diamino-N—(N-(4-(4-(2-((3-((R)-2-amino-6-guanidinohexanamido)propyl)(3-((S)-2-amino-6-guanidinohexanamido)propyl)amino)ethoxy)phenyl)butyl)carbamimidoyl)-6-chloropyrazine-2-carboxamide-Compound90

Preparation of Compound 85

A solution of amine 83 (1.19 g, 3.47 mmol) and compound 84 (1.90 g, 3.49mmol) in MeOH (60 mL) was charged with NaCNBH₃ (510 mg, 7.00 mmol) andAcOH (630 mg, 10.5 mmol). The reaction mixture was stirred at roomtemperature for 16 h. After the solvent was removed, the residue waswashed with 1 N Na₂CO₃ (100 mL), dissolved in CH₂Cl₂ (200 mL), andwashed with water (100 mL) and brine (100 mL). The organic layer wasevaporated to dryness and the residue was purified by columnchromatography (silica gel, 20:1 CH₂Cl₂/MeOH) to afford compound 85(1.58 g, 52%) as a colorless oil: ¹H NMR (300 MHz, DMSO-d₆) δ 8.27 (brs, 1H), 7.83 (br s, 1H), 7.38-7.26 (m, 5H), 7.06 (d, J=8.4 Hz, 2H), 6.82(d, J=8.4 Hz, 2H), 6.77 (br s, 1H), 5.76 (br s, 3H), 4.99 (s, 2H), 3.94(br s, 2H), 3.83 (br s, 1H), 3.27-3.22 (m, 2H), 3.16-2.98 (m, 4H), 2.83(br s, 2H), 1.55-1.48 (m, 5H), 1.46 (s, 11H), 1.38 (s, 10H), 1.36 (s,10H).

Preparation of Compound 87

A solution of compound 85 (1.18 g, 1.36 mmol) and compound 86 (1.10 g,2.03 mmol) in MeOH (20 mL) was charged with NaCNBH₃ (297 mg, 4.08 mmol)and AcOH (326 mg, 5.44 mmol). The reaction mixture was stirred at roomtemperature for 16 h. Additional compound 86 (1.10 g, 2.03 mmol),NaCNBH₃ (297 mg, 4.08 mmol), and AcOH (326 mg, 5.44 mmol) were added.The reaction mixture continued to stir at room temperature for 16 h.After the solvent was removed, the residue was washed with 1 N Na₂CO₃(100 mL), dissolved in CH₂Cl₂ (200 mL), and washed with water (100 mL)and brine (100 mL). The organic layer was evaporated to dryness and theresidue was purified by column chromatography (silica gel, 20:1CH₂Cl₂/MeOH) to afford compound 87 (2.12 g, mixture) as a colorless oil,which was used directly in the next step.

Preparation of Compound 88

A suspension of compound 87 (2.12 g, mixture) and 10% Pd/C (1.00 g) inEtOH (30 mL) was subjected to hydrogenation conditions (1 atm) for 4 hat room temperature. The reaction mixture was filtered through celiteand washed with EtOH. The filtrate was concentrated and purified bycolumn chromatography (silica gel, 20:1 CH₂Cl₂/MeOH, 8:1:0.1CHCl₃/MeOH/NH₄OH) to afford compound 88 (732 mg, 43% over 2 steps) as acolorless oil: ¹H NMR (300 MHz, CD₃OD) δ 7.09 (d, J=8.4 Hz, 2H), 6.84(d, J=8.4 Hz, 2H), 4.05-3.95 (m, 4H), 3.27-3.21 (m, 4H), 2.88-2.83 (m,4H), 2.62-2.56 (m, 6H), 1.77-1.55 (m, 15H), 1.51 (s, 18H), 1.46 (s,18H), 1.43 (s, 18H).

Preparation of Compound 89

A solution of amine 88 (710 mg, 0.562 mmol) and methyl3,5-diamino-6-chloropyrazine-2-carbonylcarbamimidothioate hydroiodicacid salt (7, 261 mg, 0.675 mmol) in t-BuOH (15 mL) was charged withDIPEA (359 mg, 2.81 mmol) at room temperature. The reaction mixture washeated at 70° C. for 2 h, cooled to room temperature, and concentratedin vacuum. The residue was purified by column chromatography (silicagel, 10:1 CH₂Cl₂/MeOH, 10:1:0.1 CHCl₃/MeOH/NH₄OH) to afford compound 89(350 mg, 43%) as a yellow solid: ¹H NMR (300 MHz, CD₃OD) δ 7.10 (d,J=8.6 Hz, 2H), 6.84 (d, J=8.6 Hz, 2H), 4.08-3.98 (m, 4H), 3.76-3.67 (m,1H), 2.86 (br s, 3H), 2.63-2.58 (m, 6H), 1.73-1.64 (m, 10H), 1.62-1.56(m, 4H), 1.51 (s, 19H), 1.46 (s, 18H), 1.42 (s, 18H).

Preparation of The Hydrochloride Salt of3,5-diamino-N—(N-(4-(4-(2-((3-((R)-2-amino-6-guanidinohexanamido)propyl)(3-((S)-2-amino-6-guanidinohexanamido)propyl)amino)ethoxy)phenyl)butyl)carbamimidoyl)-6-chloropyrazine-2-carboxamide-Compound90

A solution of compound 89 (230 mg, 0.156 mmol) in EtOH (1.0 mL) wascharged with 4 N aqueous HCl (10 mL) at room temperature and thereaction mixture was stirred for 4 h at room temperature. The reactionmixture was concentrated in vacuum and the residue was purified byreverse-phase column chromatography and lyophilized to affordhydrochloric acid salt 90 (59 mg, 35%) as a yellow hygroscopic solid: ¹HNMR (400 MHz, D₂O) δ 7.21 (d, J=8.4 Hz, 2H), 6.91 (d, J=8.4 Hz, 2H),4.19 (br s, 2H), 3.77 (t, J=6.4 Hz, 2H), 3.46 (br s, 2H), 3.25-3.21 (m,8H), 3.13 (br s, 4H), 3.02 (t, J=7.0 Hz, 4H), 2.53 (br s, 2H), 1.93-1.86(m, 4H), 1.75-1.70 (m, 4H), 1.60 (br s, 4H), 1.49-1.42 (m, 4H),1.30-1.22 (m, 4H). HRMS calculated for C₃₈H₆₈ClN₁₈O₄ [M+H]⁺, 875.5354.found 875.5372. Elemental analysis: % calculated C, 41.71; H, 6.72; N,23.04. found C, 38.03; H, 5.80; N, 20.40.

Preparation The Hydrochloride Salt of(S)-3,5-diamino-N—(N-(4-(4-(2-(3-(2-amino-6-guanidinohexanamido)propylamino)ethoxy)phenyl)butyl)carbamimidoyl)-6-chloropyrazine-2-carboxamide-Compound94

Preparation of Compound 91

A solution of compound 85 (400 mg, 0.460 mmol) in THF (6.0 mL), MeOH(6.0 mL), and water (2.0 mL) was charged with NaHCO₃ (116 mg, 1.38 mmol)and Boc₂O (120 mg, 0.550 mmol). The reaction mixture was stirred for 3 hat room temperature. After the solvent was removed, the residue waspartitioned between CH₂Cl₂ (20 mL) and water (10 mL). The aqueous layerwas separated and extracted with CH₂Cl₂ (20 mL). The combined organicextracts were dried over Na₂SO₄, concentrated, and purified by columnchromatography (silica gel, 10:1 CH₂Cl₂/MeOH), to afford compound 91(379 mg, 85%) as a white solid: ¹H NMR (300 MHz, CD₃OD) δ 7.32-7.28 (m,5H), 7.05 (d, J=8.6 Hz, 2H), 6.80 (d, J=8.6 Hz, 2H), 5.05 (s, 2H),4.10-4.04 (m, 2H), 3.95 (br s, 1H), 3.57 (t, J=5.6 Hz, 2H), 3.23-3.09(m, 4H), 2.54 (t, J=7.2 Hz, 2H), 1.77 (br s, 2H), 1.62-1.52 (m, 4H),1.51 (s, 11H), 1.45-1.42 (m, 28H).

Preparation of Compound 92

A suspension of compound 91 (375 mg, 0.387 mmol) and 10% Pd/C (200 mg)in EtOH (15 mL) was subjected to hydrogenation conditions (1 atm) for 2h at room temperature. The reaction mixture was filtered through celiteand washed with EtOH. The filtrate was concentrated to afford compound92 (297 mg, 92%) as a white solid: ¹H NMR (300 MHz, CD₃OD) δ 7.09 (d,J=8.4 Hz, 2H), 6.82 (d, J=8.4 Hz, 2H), 4.05 (br s, 2H), 3.99-3.94 (m,1H), 3.57 (t, J=5.2 Hz, 2H), 3.25-3.19 (m, 2H), 2.71 (t, J=6.8 Hz, 2H),2.57 (t, J=7.2 Hz, 2H), 1.76 (br s, 3H), 1.65-1.54 (m, 5H), 1.51 (s,10H), 1.47-1.45 (m, 18H), 1.42 (s, 10H).

Preparation of Compound 93

A solution of amine 92 (295 mg, 0.353 mmol) and methyl3,5-diamino-6-chloropyrazine-2-carbonylcarbamimidothioate hydroiodicacid salt (7, 165 mg, 0.424 mmol) in t-BuOH (20 mL) was charged withDIPEA (227 mg, 1.76 mmol) at room temperature. The reaction mixture washeated at 70° C. for 2 h, cooled to room temperature, and concentratedin vacuum. The residue was purified by column chromatography (silicagel, 10:1 CH₂Cl₂/MeOH, 10:1:0.1 CHCl₃/MeOH/NH₄OH) to afford compound 93(244 mg, 66%) as a yellow solid: ¹H NMR (300 MHz, CD₃OD) δ 7.10 (d,J=8.4 Hz, 2H), 6.82 (d, J=8.4 Hz, 2H), 4.07-4.04 (m, 2H), 3.98-3.93 (m,1H), 3.57 (t, J=5.6 Hz, 2H), 3.25-3.17 (m, 3H), 2.62 (br s, 2H),1.77-1.56 (m, 8H), 1.51 (s, 9H), 1.46 (s, 18H), 1.42 (s, 10H).

Preparation The Hydrochloride Salt of(S)-3,5-diamino-N—(N-(4-(4-(2-(3-(2-amino-6-guanidinohexanamido)propylamino)ethoxy)phenyl)butyl)carbamimidoyl)-6-chloropyrazine-2-carboxamide-Compound94

A solution of compound 73 (238 mg, 0.227 mmol) in EtOH (3.0 mL) wascharged with 4 N aqueous HCl (10 mL) at room temperature and thereaction mixture was stirred for 4 h at room temperature. The reactionmixture was concentrated in vacuum, and the residue was purified byreverse-phase column chromatography and lyophilized to affordhydrochloric acid salt 74 (96 mg, 53%) as a yellow hygroscopic solid: ¹HNMR (400 MHz, D₂O) δ 7.20 (d, J=8.6 Hz, 2H), 6.91 (d, J=8.6 Hz, 2H),4.21 (br s, 2H), 3.82 (br s, 1H), 3.42 (t, J=4.8 Hz, 2H), 3.34-3.28 (m,4H), 3.13-3.08 (m, 4H), 2.59 (br s, 2H), 1.92 (t, J=7.6 Hz, 2H), 1.78(br s, 2H), 1.66 (br s, 4H), 1.56-1.50 (m, 2H), 1.37-1.31 (m, 2H). HRMScalculated for C₂₈H₄₇ClN₁₃O₃ [M+H]⁺, 648.3608. found 648.3619. Elementalanalysis: % calculated C, 42.35; H, 6.35; N, 22.32. found C, 37.84; H,6.57; N, 20.29.

Preparation of The Hydrochloride Salt of(S,S,2S,2′S)—N,N′-(3,3′-(2-(4-(4-(3-(3,5-diamino-6-chloropyrazine-2-carbonyl)guanidino)butyl)phenoxy)ethylazanediyl)bis(propane-3,1-diyl))bis(2-amino-6-((S)-2-amino-6-((S)-2-amino-6-guanidinohexanamido)hexanamido)hexanamide)

Preparation of Compound 95

A stirred solution of amino acid 11 (4.00 g, 8.19 mmol) in CH₂Cl₂ (100mL) was charged with EEDQ (2.42 g, 9.83 mmol) and NMM (2.50 g, 24.5mmol). The reaction mixture was stirred at room temperature for 10 minand amine 53 (2.12 g, 8.19 mmol) was added. The resulting mixture wasstirred at room temperature for 16 h. The solvent was removed and theresidue was purified by column chromatography (silica gel, 10:1CH₂Cl₂/EtOAc, 10:1 CH₂Cl₂/MeOH) to afford amide 75 (3.80 g, 74%) as awhite solid: ¹H NMR (300 MHz, CDCl₃) δ 11.48 (s, 1H), 8.32 (t, J=5.2 Hz,1H), 6.36 (br s, 1H), 5.23-5.14 (m, 2H), 4.28-4.19 (m, 1H), 4.06-3.97(m, 1H), 3.73 (s, 3H), 3.42-3.15 (m, 4H), 1.90-1.69 (m, 6H), 1.67-1.55(m, 4H), 1.49 (s, 18H), 1.47 (s, 9H), 1.43 (s, 9H), 1.41-1.36 (m, 4H).

Preparation of Compound 96

A solution of methyl ester 95 (3.80 g, 5.20 mmol) in THF/H₂O (50 mL/10mL) was charged with NaOH (416 mg, 10.41 mmol) and the reaction mixturewas stirred at room temperature for 3 h. After completion of thereaction, the mixture was concentrated under reduced pressure and the pHwas adjusted to 9 with 1 N NaOH. The aqueous solution was washed withEtOAc (2×150 mL) and the pH was adjusted to 5. The suspension waspartitioned between CH₂Cl₂ (200 mL) and water (200 mL). The aqueouslayer was separated and extracted with CH₂Cl₂ (2×200 mL). The combinedorganic extracts were dried over Na₂SO₄ and concentrated to affordcompound 96 (crude, 3.30 g, 89%) as a white solid, which was useddirectly in the next step.

Preparation of Compound 97

A stirred solution of amino acid 96 (crude, 3.30 g, 4.60 mmol) in CH₂Cl₂(100 mL) was charged with EEDQ (1.36 g, 5.53 mmol) and NMM (1.40 g, 13.8mmol). The reaction mixture was stirred at room temperature for 10 minand amine 53 (1.20 g, 4.60 mmol) was added. The resulting mixture wasstirred at room temperature for 16 h. The solvent was removed and theresidue was purified by column chromatography (silica gel, 10:1CH₂Cl₂/EtOAc, 10:1 CH₂Cl₂/MeOH) to afford compound 77 (3.51 g, 80%) as awhite solid: ¹H NMR (300 MHz, CDCl₃) δ 11.47 (s, 1H), 8.30 (t, J=4.8 Hz,1H), 6.40 (br s, 2H), 5.41-5.32 (m, 1H), 5.25-5.12 (m, 2H), 4.23 (br s,1H), 4.11-3.96 (m, 3H), 3.42-3.36 (m, 2H), 3.25-3.15 (m, 4H), 1.87-1.74(m, 4H), 1.54-1.46 (m, 23H), 1.46-1.40 (m, 30H), 1.39-1.31 (m, 6H).

Preparation of Compound 98

A solution of methyl ester 97 (3.51 g, 3.66 mmol) in THF/H₂O (50 mL/10mL) was charged with NaOH (293 mg, 7.32 mmol) and the reaction mixturewas stirred at room temperature for 3 h. After completion of thereaction, the mixture was concentrated under reduced pressure and the pHwas adjusted to 9 with 1 N NaOH. The aqueous solution was washed withEtOAc (2×150 mL) and the pH was adjusted to 5. The suspension waspartitioned between CH₂Cl₂ (200 mL) and water (200 mL). The aqueouslayer was separated and extracted with CH₂Cl₂ (2×200 mL). The combinedorganic extracts were dried over Na₂SO₄ and concentrated to affordcompound 98 (3.00 g, 88%) as a white solid: ¹H NMR (400 MHz, CDCl₃) δ8.34 (br s, 1H), 6.93 (br s, 1H), 6.61 (br s, 1H), 5.51-5.40 (m, 3H),4.28 (br s, 1H), 4.14-4.04 (m, 3H), 3.76-3.16 (m, 6H), 1.87-1.75 (m,7H), 1.65-1.51 (m, 5H), 1.49 (s, 9H), 1.48 (s, 9H), 1.44 (s, 9H), 1.42(s, 18H), 1.39-1.26 (m, 6H).

Preparation of Compound 99

A stirred solution of compound 4 (free base, 500 mg, 1.09 mmol) inCH₂Cl₂ (50 mL) was charged with EEDQ (1.21 g, 4.93 mmol) and amino acid98 (2.57 g, 2.72 mmol). The resulting mixture was stirred at roomtemperature for 16 h. Additional amino acid 98 (515 mg, 0.545 mmol) andEEDQ (270 mg, 1.09 mmol) were added. The resulting mixture was stirredat room temperature for 6 h. The solvent was removed and the residue waspurified by column chromatography (silica gel, 10:1 CH₂Cl₂/EtOAc, 10:1CH₂Cl₂/MeOH) to afford amide 99 (1.42 g, 70%) as a white solid: ¹H NMR(400 MHz, CD₃OD) δ 7.33-7.27 (m, 4H), 7.06 (d, J=8.2 Hz, 2H), 6.82 (d,J=8.2 Hz, 2H), 5.05 (s, 2H), 4.05-3.96 (m, 10H), 3.68 (t, J=4.2 Hz, 1H),3.34 (t, J=7.0 Hz, 4H), 3.24-3.10 (m, 16H), 2.88-2.83 (m, 2H), 2.61-2.52(m, 6H), 2.43 (br s, 1H), 1.74-1.66 (m, 12H), 1.63-1.54 (m, 14H), 1.51(s, 25H), 1.46 (s, 25H), 1.44 (s, 20H), 1.43 (s, 20H), 1.42 (s, 20H).

Preparation of Compound 100

A stirred solution of compound 99 (300 mg 0.129 mmol) in t-BuOH (10 mL)and THF (2.0 mL) was charged with 10% Pd/C (150 mg) and the mixture wassubjected to hydrogenation conditions (1 atm) for 16 h at roomtemperature. After completion of the reaction, the mixture was filteredthrough celite and washed with THF. The filtrate was concentrated underreduced pressure to afford compound 100 (260 mg, 92%) as a brown solid:¹H NMR (400 MHz, CD₃OD): δ 7.09 (d, J=8.8 Hz, 2H), 6.84 (d, J=8.8 Hz,2H), 4.03-3.96 (m, 8H), 3.35-3.12 (m, 14H), 2.90-2.83 (m, 4H), 2.60-2.57(m, 6H), 1.70-1.31 (m, 154H).

Preparation of Compound 101

A stirred solution of compound 100 (1.43 g, 0.650 mmol) was charged withmethyl (3,5-diamino-6-chloropyrazine-2-carbonyl)carbamimidothioatehydroiodide 7 (253 mg, 0.650 mmol) and NMM (332 mg, 3.28 mmol) in t-BuOH(60 mL) and THF (12 mL). The reaction mixture was stirred for 4 h at 60°C. and at 70° C. for 1 h. After completion of the reaction, the mixturewas concentrated under reduced pressure and purified by columnchromatography (silica gel, 10:1 CH₂Cl₂/MeOH, 5:1:0.1 CHCl₃/MeOH/NH₄OH)to afford compound 101 (1.24 g, 79%) as a yellow solid: ¹H NMR (300 MHz,CD₃OD): δ 7.14 (d, J=7.8 Hz, 2H), 6.90 (d, J=7.8 Hz, 2H), 4.20 (br s,2H), 3.98-3.89 (m, 6H), 3.79-3.74 (m, 2H), 3.34-3.07 (m, 12H), 2.84-2.77(m, 4H), 2.66-2.56 (m, 6H), 1.70-1.31 (m, 154H).

Preparation of The Hydrochloride Salt of(S,S,2S,2′S)—N,N′-(3,3′-(2-(4-(4-(3-(3,5-diamino-6-chloropyrazine-2-carbonyl)guanidino)butyl)phenoxy)ethylazanediyl)bis(propane-3,1-diyl))bis(2-amino-6-((S)-2-amino-6-((S)-2-amino-6-guanidinohexanamido)hexanamido)hexanamide)-Compound102

A solution of compound 81 (1.40 g, 0.50 mmol) in EtOH (30 mL) wascharged with 4 N aqueous HCl (100 mL) and the reaction mixture wasstirred for 4 h at room temperature. The reaction mixture wasconcentrated and fresh 4 N aqueous HCl was added. After stirring for 4 hat room temperature, the reaction mixture was concentrated in vacuum andthe residue was purified by reverse-phase column chromatography andlyophilized to afford hydrochloric acid salt 82 (450 mg, 62%) as ayellow hygroscopic solid: ¹H NMR (400 MHz, D₂O) δ 7.22 (d, J=8.8 Hz,2H), 6.92 (d, J=8.8 Hz, 2H), 4.29 (br s, 2H), 3.91-3.88 (m, 6H), 3.60(br s, 2H), 3.39-3.11 (m, 23H), 2.60 (br s, 2H), 2.01-1.97 (m, 4H),1.86-1.78 (m, 13H), 1.67-1.46 (m, 17H), 1.40-1.32 (m, 12H).

Preparation of The Hydrochloride Salt of(S,2S,2′S)—N,N′-(3,3′-(2-(4-(4-(3-(3,5-diamino-6-chloropyrazine-2-carbonyl)guanidino)butyl)phenoxy)ethylazanediyl)bis(propane-3,1-diyl))bis(2-amino-6-((S)-2-amino-6-guanidinohexanamido)hexanamide)-Compound106

Preparation of Compound 103

A solution of amino acid 96 (100 mg, 0.139 mmol) in CH₂Cl₂ (5.0 mL) wascharged with EEDQ (84 mg, 0.280 mmol) and compound 4 (free base, 32.0mg, 0.0701 mmol). The reaction mixture was stirred at room temperaturefor 16 h. The solvent was removed and the residue was purified by columnchromatography (silica gel, 10:1 CH₂Cl₂/MeOH) to afford compound 103(78.0 mg, 61%) as a white solid: ¹H NMR (300 MHz, CD₃OD) δ 7.33-7.28 (m,5H), 7.07 (d, J=8.4 Hz, 2H), 6.84 (d, J=8.4 Hz, 2H), 5.05 (s, 2H),4.08-3.95 (m, 6H), 3.29-3.21 (m, 4H), 3.19-3.06 (m, 7H), 2.95 (br s,2H), 2.68 (br s, 3H), 2.54 (t, J=6.8 Hz, 2H), 1.77-1.55 (m, 17H), 1.51(s, 21H), 1.46 (s, 18H), 1.43 (s, 18H), 1.42 (s, 18H).

Preparation of Compound 104

A suspension of compound 103 (736 mg, 0.397 mmol) and 10% Pd/C (380 mg)in EtOH (15 mL) was subjected to hydrogenation conditions (1 atm) for 6h at room temperature. The reaction mixture was filtered through celiteand washed with EtOH. The filtrate was concentrated and precipitatedfrom MTBE/hexanes to afford compound 104 (627 mg, 92%) as a white solid:¹H NMR (300 MHz, CD₃OD) δ 7.09 (d, J=8.6 Hz, 2H), 6.84 (d, J=8.6 Hz,2H), 4.05-3.93 (m, 6H), 3.27-3.06 (m, 6H), 2.85 (t, J=7.6 Hz, 4H), 2.58(br s, 6H), 1.73-1.54 (m, 20H), 1.51 (s, 21H), 1.46 (s, 19H), 1.43 (s,22H), 1.42 (s, 18H).

Preparation of Compound 105

A solution of amine 104 (624 mg, 0.363 mmol) and methyl3,5-diamino-6-chloropyrazine-2-carbonylcarbamimidothioate hydroiodicacid salt (7, 169 mg, 0.436 mmol) in t-BuOH (15 mL) was charged withDIPEA (235 mg, 1.81 mmol) at room temperature. The reaction mixture washeated at 70° C. for 2 h, cooled to room temperature, and concentratedin vacuum. The residue was purified by column chromatography (silicagel, 20:1 CH₂Cl₂/MeOH) to afford compound 105 (350 mg, 50%) as a yellowsolid: ¹H NMR (300 MHz, CD₃OD) δ 7.10 (d, J=8.6 Hz, 2H), 6.84 (d, J=8.6Hz, 2H), 4.02-3.95 (m, 6H), 3.23-3.06 (m, 5H), 2.84 (br s, 2H), 2.58 (brs, 6H), 1.71-1.54 (m, 20H), 1.51 (s, 21H), 1.46 (s, 20H), 1.43 (s, 22H),1.42 (s, 18H).

Preparation of The Hydrochloride Salt of(S,2S,2′S)—N,N′-(3,3′-(2-(4-(4-(3-(3,5-diamino-6-chloropyrazine-2-carbonyl)guanidino)butyl)phenoxy)ethylazanediyl)bis(propane-3,1-diyl))bis(2-amino-6-((S)-2-amino-6-guanidinohexanamido)hexanamide)-Compound106

A solution of compound 105 (347 mg, 0.179 mmol) in CH₂Cl₂ (10 mL) wascharged with TFA (5.0 mL) at room temperature and the reaction mixturewas stirred for 2 h at room temperature. The reaction mixture wasconcentrated in vacuum and twice azeotroped with 1 N aqueous HCl. Theresidue was purified by reverse-phase column chromatography andlyophilized to afford hydrochloric acid salt 106 (155 mg, 61%) as ayellow hygroscopic solid: ¹H NMR (400 MHz, D₂O) δ 7.22 (d, J=8.4 Hz,2H), 6.91 (d, J=8.4 Hz, 2H), 4.28 (br s, 2H), 3.90-3.86 (m, 4H), 3.60(br s, 2H), 3.35-3.11 (m, 18H), 2.60 (br s, 2H), 2.00-1.95 (m, 4H),1.84-1.79 (m, 8H), 1.67 (br s, 4H), 1.57-1.47 (m, 8H), 1.37-1.32 (m,8H). HRMS calculated for C₅₀H₉₂ClN₂₂O₆ [M+H]⁺, 1131.7253; found1131.7297.

Preparation of The Hydrochloride Salt of(2R,2′R)—N,N′-(3,3′-(2-(6-(4-(3-(3,5-diamino-6-chloropyrazine-2-carbonyl)guanidino)butyl)naphthalen-2-yloxy)ethylazanediyl)bis(propane-3,1-diyl))bis(2-amino-6-guanidinohexanamide)-Compound116

Preparation of Compound 109

A stirred solution of compound 107 (5.00 g, 22.5 mmol) in dry CH₂Cl₂(100 mL) was charged with compound 108 (4.30 g, 27.1 mmol), Ph₃P (7.10g, 27.1 mmol), and DIAD (5.40 g, 27.1 mmol) at 0° C. The reactionmixture was warmed to room temperature and stirred for 4 h. Aftercompletion of the reaction, the mixture was diluted with CH₂C₂ andwashed with 1 N NaHCO₃, water, and brine. The organic layer wasconcentrated under reduced pressure and purified by columnchromatography (silica gel, 80:20 hexanes/EtOAc) to afford compound 109(5.50 g, 67%) as an off-white solid: ESI-MS m/z 366 [C₁₇H₂₀BrNO₃+H]⁺.

Preparation of Compound 110

Compound 109 (5.50 g, 15.1 mmol) was dissolved in 4 N HCl in dioxane (50mL) at room temperature and the solution was stirred for 3 h. Afterconcentration, the residue was suspended in MTBE (50 mL) and stirred for0.5 h. The solid was filtered out to afford hydrochloric acid salt 110(3.20 g, 82%) as a white solid: ¹H NMR (300 MHz, CD₃OD) δ 7.99 (br s,1H), 7.77-7.70 (m, 2H), 7.55-7.51 (m, 1H), 7.33-7.26 (m, 2H), 4.36 (t,J=4.8 Hz, 2H), 3.43 (t, J=4.8 Hz, 2H).

Preparation of Compound 112

A stirred solution of compound 110 (3.20 g, 12.1) in anhydrous CH₃CN(150 mL) was charged with TEA (4.8 g, 48.3 mmol), 10% (t-Bu)₃P inhexanes (0.48 g, 2.42 mmol), compound 111 (3.68 g, 18.1 mmol), and CuI(114 mg, 0.6 mmol) at room temperature. The resulting mixture wasdegassed with argon for 10 min and Pd(PPh₃)₄ (1.40 g, 1.21 mmol) wasadded rapidly in one portion. After degassing with argon for 5 min, themixture was refluxed for 4 h. The reaction mixture was concentratedunder vacuum and the residue was purified by column chromatography(silica gel, 80:20 hexanes/EtOAc) to afford compound 112 (2.80 g, 61%)as a brown solid: ¹H NMR (400 MHz, CD₃OD) δ 7.80 (br s, 1H), 7.67 (t,J=8.2 Hz, 2H), 7.37-7.16 (m, 8H), 5.09 (s, 2H), 4.14 (t, J=5.2 Hz, 2H),3.37 (t, J=6.8 Hz, 2H), 3.09 (br s, 2H), 2.60 (t, J=6.8 Hz, 2H).

Preparation of Compound 113

A stirred solution of compound 112 (1.00 g, 2.57 mmol) in MeOH (80 mL)was charged with NaCNBH₃ (480 mg, 7.71 mmol), acetic acid (0.6 g, 10.28mmol), and aldehyde 86 (3.50 g, 6.44 mmol). The reaction mixture wasstirred at room temperature for 6 h. After completion of the reaction,the mixture was concentrated under reduced pressure. The residue waspartitioned between EtOAc (300 mL) and saturated NaHCO₃ (200 mL). Theaqueous layer was separated and extracted with EtOAc (2 t 300 mL). Thecombined organic extracts were dried over Na₂SO₄, concentrated, andpurified by column chromatography (silica gel, 10:1 CH₂Cl₂/CH₃OH) toafford compound 113 (2.50 g, 68%) as a yellow solid: ¹H NMR (400 MHz,DMSO-d₆) δ 11.49 (s, 2H), 8.31-8.22 (m, 3H), 7.79-7.73 (m, 4H), 7.52 (t,J=5.8 Hz, 1H), 7.38-7.28 (m, 7H), 7.17-7.14 (m, 1H), 6.73 (d, J=8.2 Hz,2H), 5.04 (s, 2H), 4.11 (t, J=5.8 Hz, 2H), 3.83 (t, J=5.8 Hz, 2H),3.41-3.36 (m, 1H), 3.27-3.21 (m, 7H), 3.12-3.06 (m, 5H), 2.82 (t, J=5.6Hz, 2H), 2.58 (t, J=6.8 Hz, 2H), 1.54-1.50 (m, 8H), 1.47-1.43 (m, 30H),1.39-1.33 (m, 48H), 1.29-1.23 (m, 6H).

Preparation of Compound 114

A stirred solution of compound 113 (2.50 g, 1.73 mmol) in EtOH (50 mL)was charged with 10% Pd/C (250 mg) and was subjected to hydrogenationconditions (I atm) for 12 h at room temperature. The reaction mixturewas filtered through celite and washed with EtOH. The filtrate wasconcentrated under reduced pressure and purified by columnchromatography (silica gel, 10:1 CH₂Cl₂/CH₃OH) to afford compound 114(1.20 g, 55%) as a brown solid: ¹H NMR (400 MHz, CD₃OD) δ 7.69-7.66 (m,2H), 7.55 (br s, 1H), 7.30-7.26 (m, 1H), 7.21 (br s, 1H), 7.12-7.09 (m,1H), 4.17 (t, J=5.6 Hz, 2H), 3.98 (br s, 2H), 3.37-3.32 (m, 2H), 2.93(t, J=5.6 Hz, 2H), 2.79-2.71 (m, 4H), 2.62 (t, J=6.6 Hz, 4H), 1.76-1.65(m, 9H), 1.63-1.55 (m, 6H), 1.52-1.50 (m, 25H), 1.46-1.45 (m, 23H),1.43-1.42 (m, 25H).

Preparation of Compound 115

A stirred solution of compound 114 (240 mg, 0.18 mmol) in t-BuOH (5 mL)and THF (1 mL) was charged with methyl(3,5-diamino-6-chloropyrazine-2-carbonyl)carbamimidothioate hydroiodide7 (71 mg, 0.18 mmol) and NMM (0.9 g, 0.9 mmol). The reaction mixture wasstirred for 4 h at 60° C. After concentration, the residue was purifiedby column chromatography (silica gel, 10:1 CH₂Cl₂/MeOH, 5:1:0.1CHCl₃/MeOH/NH₄OH) to afford compound 115 (140 mg, 46%) as a yellowsolid: ¹H NMR (400 MHz, CD₃OD) δ 7.69-7.62 (m, 2H), 7.56 (br s, 1H),7.36-7.29 (m, 1H), 7.20 (br s, 1H), 7.11-7.08 (m, 1H), 4.17 (t, J=5.6Hz, 2H), 3.97 (br s, 2H), 3.69-3.63 (m, 1H), 2.93 (t, J=5.4 Hz, 2H),2.81 (t, J=7.2 Hz, 2H), 2.62 (t, J=6.8 Hz, 4H), 1.86-1.79 (m, 2H),1.74-1.69 (m, 8H), 1.60-1.29 (m, 66H).

Preparation of The Hydrochloride Salt of(2R,2′R)—N,N′-(3,3′-(2-(6-(4-(3-(3,5-diamino-6-chloropyrazine-2-carbonyl)guanidino)butyl)naphthalen-2-yloxy)ethylazanediyl)bis(propane-3,1-diyl))bis(2-amino-6-guanidinohexanamide)-Compound116

A solution of compound 115 (140 mg, 0.091 mmol) in EtOH (1.0 mL) wascharged with 4 N aqueous HCl (5.0 mL) and the reaction mixture wasstirred for 3 h at room temperature. After concentration, the residuewas purified by reverse-phase column chromatography and lyophilized toafford hydrochloric acid salt 116 (40 mg, 47%) as a yellow hygroscopicsolid: ¹H NMR (400 MHz, D₂O) δ 7.72-7.65 (m, 3H), 7.39 (d, J=9.2 Hz,1H), 7.12 (br s, 1H), 6.99-6.96 (m, 1H), 4.31 (br s, 2H), 3.79-3.74 (m,2H), 3.61-3.57 (m, 2H), 3.33-3.20 (m, 10H), 2.90 (t, J=7.4 Hz, 4H), 2.74(t, J=5.2 Hz, 2H), 2.10-1.94 (m, 4H), 1.85-1.65 (m, 9H), 1.38-1.31 (m,4H), 1.25-1.19 (m, 4H).

Preparation of Hydrochloride Salt of(2R,2′R)—N,N′-(3,3′-(2-(4-(4-(3-(3,5-diamino-6-chloropyrazine-2-carbonyl)guanidino)butyl)naphthalen-1-yloxy)ethylazanediyl)bis(propane-3,1-diyl))bis(2-amino-6-guanidinohexanamide)-Compound124

Preparation of Compound 118

A stirred solution of compound 117 (5.00 g, 22.5 mmol) in dry CH₂Cl₂(100 mL) was charged with compound 108 (4.30 g, 27.1 mmol), Ph₃P (7.10g, 27.1 mmol), and DIAD (5.40 g, 27.1 mmol) at 0° C. The reactionmixture was warmed to room temperature and stirred for 4 h. Aftercompletion of the reaction, the mixture was diluted with CH₂Cl₂ andwashed with 1 N NaHCO₃, water, and brine. The organic layer wasconcentrated under reduced pressure and purified by columnchromatography (silica gel, 80:20 hexanes/EtOAc) to afford compound 118(5.40 g, 66%) as an off-white solid: ESI-MS m/z 366 [C₁₇H₂₀BrNO₃+H]⁺.

Preparation of Compound 119

Compound 118 (5.40 g, 14.8 mmol) was dissolved in 4 N HCl in dioxane (50mL) at room temperature and the solution was stirred for 3 h. Afterconcentration, the residue was suspended in MTBE (50 mL) and stirred for0.5 h. The solid was filtered to afford hydrochloric acid salt 119 (3.40g, 87%) as a white solid: ¹H NMR (400 MHz, CD₃OD) δ 8.46-8.44 (m, 1H),8.17-8.14 (m, 1H), 7.73-7.56 (m, 3H), 6.91 (d, J=8.2 Hz, 1H), 4.42 (t,J=5.2 Hz, 2H), 3.53 (t, J=5.2 Hz, 2H).

Preparation of Compound 120

A stirred solution of compound 119 (3.40 g, 12.8) in anhydrous CH₃CN(150 mL) was charged with TEA (5.1 g, 51.3 mmol), 10% (t-Bu)₃P inhexanes (0.51 g, 2.56 mmol), compound 111 (3.90 g, 19.2 mmol), and CuI(121 mg, 0.64 mmol) at room temperature. The resulting mixture wasdegassed with argon for 10 min and Pd(PPh₃)₄(1.48 g, 1.28 mmol) wasadded rapidly in one portion. After degassing with argon for 5 min, theresulting mixture was refluxed for 4 h. The reaction mixture wasconcentrated under vacuum and the residue was purified by columnchromatography (silica gel, 80:20 hexanes/EtOAc) to afford compound 120(3.20 g, 65%) as a brown solid: ¹H NMR (400 MHz, CD₃OD) δ 8.41-8.39 (m,1H), 8.26 (d, J=7.4 Hz, 1H), 7.57-7.50 (m, 3H), 7.34-7.24 (m, 5H), 6.92(d, J=7.8 Hz, 1H), 5.09 (s, 2H), 4.43 (t, J=5.2 Hz, 2H), 3.53 (t, J=5.2Hz, 2H), 3.43 (t, J=6.8 Hz, 2H), 2.75 (t, J=6.8 Hz, 2H).

Preparation of Compound 121

A stirred solution of compound 120 (500 mg, 1.29 mmol) in 1,2-DCE wascharged with NaBH(AcO)₃ (815 mg, 3.86 mmol) and aldehyde 86 (1.40 g,2.58 mmol). The reaction mixture was stirred at room temperature for 3h. Additional NaBH(AcO)₃ (270 mg, 1.29 mmol) and aldehyde 86 (140 mg,0.258 mmol) were added and the reaction mixture was stirred for 3 h atroom temperature. After concentration, the residue was partitionedbetween CH₂Cl₂ (300 mL) and saturated NaHCO₃ (200 mL). The aqueous layerwas separated and extracted with CH₂Cl₂ (2×100 mL). The combined organicextracts were dried over Na₂SO₄ and concentrated to afford compound 121(crude, 1.20 g) as a white solid, which was used directly in the nextstep.

Preparation of Compound 122

A stirred solution of compound 121 (crude, 1.20 g) in t-BuOH (60 mL) andTHF (12 mL) was charged with 10% Pd/C (600 mg). The suspension wassubjected to hydrogenation conditions (1 atm) for 26 h at roomtemperature. The reaction mixture was filtered through celite and washedwith THF. Fresh 10% Pd/C (600 mg) was added to the filtrate and thesuspension was subjected to hydrogenation conditions (1 atm) for 24 h.The reaction mixture was filtered through celite and washed with THF.The filtrate was concentrated under reduced pressure to afford compound122 (crude, 900 mg) as a brown solid, which was used directly in thenext step.

Preparation of Compound 123

A stirred solution of compound 122 (crude, 900 mg) in t-BuOH (50 mL) wascharged with methyl(3,5-diamino-6-chloropyrazine-2-carbonyl)carbamimidothioate hydroiodide7 (316 mg, 0.82 mmol) and NMM (1.70 g, 3.4 mmol). The reaction mixturewas stirred for 4 h at 60° C., 2 h at 65° C., and 1 h at 70° C. Afterconcentration, the residue was purified by column chromatography (silicagel, 10:1 CH₂Cl₂/MeOH, 5:1:0.1 CHCl₃/MeOH/NH₄OH) to afford compound 123(310 mg, 17% over 3 steps) as a yellow solid: ¹H NMR (300 MHz, CD₃OD) δ8.24 (d, J=7.6 Hz, 1H), 7.99 (d, J=8.4 Hz, 1H), 7.53-7.40 (m, 2H), 7.24(d, J=7.8 Hz, 1H), 6.85 (d, J=7.8 Hz, 1H), 4.23 (t, J=5.4 Hz, 2H), 3.96(br s, 2H), 3.10-3.01 (m, 4H), 2.67 (t, J=6.4 Hz, 4H), 1.82-1.68 (m,11H), 1.51 (s, 26H), 1.45 (s, 26H), 1.41 (s, 22H).

Preparation of Hydrochloride Salt of(2R,2′R)—N,N′-(3,3′-(2-(4-(4-(3-(3,5-diamino-6-chloropyrazine-2-carbonyl)guanidino)butyl)naphthalen-1-yloxy)ethylazanediyl)bis(propane-3,1-diyl))bis(2-amino-6-guanidinohexanamide)-Compound124

A solution of compound 103 (310 mg, 0.203 mmol) in EtOH (2.0 mL) wascharged with 4 N aqueous HCl (15.0 mL) and the reaction mixture wasstirred for 5 h at room temperature. The reaction mixture wasconcentrated in vacuum, and the residue was purified by reverse-phasecolumn chromatography and lyophilized to afford hydrochloric acid salt104 (95 mg, 41%) as a yellow hygroscopic solid: ¹H NMR (400 MHz, D₂O) δ8.07 (d, J=8.6 Hz, 1H), 7.97 (d, J=8.2 Hz, 1H), 7.58 (t, J=7.4 Hz, 1H),7.42 (t, J=7.4 Hz, 1H), 7.33 (d, J=7.8 Hz, 1H), 6.91 (d, J=7.8 Hz, 1H),4.50 (br s, 2H), 3.79 (t, J=6.6 Hz, 4H), 3.37-3.29 (m, 8H), 3.22 (t,J=5.8 Hz, 2H), 3.03 (t, J=6.2 Hz, 2H), 2.92 (t, J=7.2 Hz, 4H), 2.08-2.01(m, 4H), 1.88-1.64 (m, 8H), 1.38-1.31 (m, 4H), 1.24-1.18 (m, 4H).

Preparation of Hydrochloride Salt of(2R,2′R)—N,N′-(3,3′-(2-(4-(4-(3-(3,5-diamino-6-chloropyrazine-2-carbonyl)guanidino)butyl)-5,6,7,8-tetrahydronaphthalen-1-yloxy)ethylazanediyl)bis(propane-3,1-diyl))bis(2-amino-6-guanidinohexanamide)Compound 133

Preparation of Compound 126

A stirred solution of compound 125 (6.00 g, 26.4 mmol) in dry CH₂Cl₂(150 mL) was charged with compound 108 (4.68 g, 29.0 mmol), Ph₃P (8.30g, 31.6 mmol), and DIAD (6.38 g, 31.6 mmol) at 0° C. The reactionmixture was warmed to room temperature and stirred for 4 h. Aftercompletion of the reaction, the mixture was diluted with CH₂Cl₂ andwashed with 1 N NaHCO₃, water, and brine. The organic layer wasconcentrated under reduced pressure and purified by columnchromatography (silica gel, 80:20 hexanes/EtOAc) to afford compound 126(6.10 g, 63%) as a white solid: ¹H NMR (300 MHz, DMSO-d₆) δ 7.32 (d,J=8.6 Hz, 1H), 6.93 (t, J=5.4 Hz, 1H), 6.72 (d, J=8.6 Hz, 1H), 3.91 (t,J=5.8 Hz, 2H), 3.30 (t, J=5.6 Hz, 2H), 2.62-2.56 (m, 4H), 1.72-1.63 (m,4H), 1.37 (s, 9H).

Preparation of Compound 127

A stirred solution of compound 126 (4.00 g, 10.8) in anhydrous CH₃CN(150 mL) was charged with TEA (4.36 g, 43.2 mmol), 10% (t-Bu)₃P inhexanes (0.43 g, 2.16 mmol), compound 111 (3.28 g, 16.2 mmol), and CuI(102 mg, 0.54 mmol) at room temperature. The resulting mixture wasdegassed with argon for 10 min and Pd(PPh₃)₄(1.24 g, 1.08 mmol) wasadded rapidly in one portion. After degassing with argon for 5 min, theresulting mixture was refluxed for 16 h. The reaction mixture wasconcentrated under vacuum and the residue was purified by columnchromatography (silica gel, 80:20 hexanes/EtOAc) to afford compound 127(2.90 g, 54%) as a brown solid: ¹H NMR (300 MHz, CDCl₃) δ 7.36-7.30 (m,6H), 7.23-7.16 (m, 1H), 6.55 (d, J=8.4 Hz, 1H), 5.11 (s, 2H), 3.98 (t,J=4.8 Hz, 2H), 3.56-3.50 (m, 2H), 3.48-3.39 (m, 2H), 2.79 (br s, 2H),2.67-2.61 (m, 4H), 1.76-1.72 (m, 4H), 1.45 (s, 9H).

Preparation of Compound 128

A stirred solution of compound 127 (4.10 g, 8.33 mmol) in EtOH (200 mL)was charged with 10% Pd/C (410 mg) and the resulting mixture wassubjected to hydrogenation conditions (1 atm) for 24 h at roomtemperature. The reaction mixture was filtered through celite and washedwith EtOH. After concentration, the residue was dissolved in dioxane (50mL) and H₂O (50 mL). CbzCl (2.11 g, 12.4 mmol) was added dropwise atroom temperature and the reaction mixture was stirred for 4 h. Afterconcentration, the residue was dissolved in CH₂Cl₂ and washed with 1 NNaHCO₃, water, and brine. The organic layer was concentrated to affordcompound 128 (crude, 2.50 g) as a brown solid, which was used directlyin the next step.

Preparation of Compound 129

Compound 128 (crude, 2.50 g) was dissolved in 4 N HCl in dioxane (50 mL)at room temperature and the solution was stirred for 3 h. Afterconcentration, the residue was neutralized with 1 N Na₂CO₃ and extractedwith CH₂Cl₂. The organic layer was concentrated and purified by columnchromatography (silica gel, 10:1 CH₂Cl₂/MeOH) to afford compound 129(1.10 g, 34% over 3 steps) as a brown oil: ¹H NMR (300 MHz, CDCl₃) δ7.36-7.26 (m, 5H), 6.89 (d, J=8.2 Hz, 1H), 6.60 (d, J=8.2 Hz, 1H), 5.11(s, 2H), 3.94 (t, J=5.2 Hz, 2H), 3.28-3.21 (m, 2H), 3.07 (t, J=5.2 Hz,2H), 2.68-2.64 (m, 4H), 2.54-2.51 (m, 2H), 1.78-1.73 (m, 4H), 1.58-1.54(m, 4H).

Preparation of Compound 130

A stirred solution of compound 129 (1.00 g, 2.52 mmol) in 1,2-DCE (80mL) was charged with NaBH(AcO)₃ (1.59 g, 7.57 mmol) and aldehyde 86(2.73 g, 5.04 mmol). The reaction mixture was stirred at roomtemperature for 3 h. Additional NaBH(AcO)₃ (530 mg, 2.52 mmol) andaldehyde 86 (820 mg, 1.51 mmol) were added and the reaction mixture wasstirred for 3 h at room temperature. After concentration, the residuewas partitioned between CH₂Cl₂ (300 mL) and saturated NaHCO₃ (200 mL).The aqueous layer was separated and extracted with CH₂Cl₂ (2×100 mL).The combined organic extracts were dried over Na₂SO₄ and concentrated toafford compound 130 (crude, 1.90 g), which was used directly in the nextstep.

Preparation of Compound 131

A stirred solution of compound 130 (crude, 2.10 g) in t-BuOH (60 mL) andTHF (20 mL) was charged with 10% Pd/C (1.10 g). The suspension wassubjected to hydrogenation conditions (1 atm) for 16 h at roomtemperature. The reaction mixture was filtered through celite and washedwith THF. The filtrate was concentrated under reduced pressure to affordcompound 131 (1.20 g crude) as a brown solid, which was used directly inthe next step.

Preparation of Compound 132

A stirred solution of compound 131 (400 mg, 0.303 mmol) in t-BuOH (20mL) and THF (4.0 mL) was charged with methyl(3,5-diamino-6-chloropyrazine-2-carbonyl)carbamimidothioate hydroiodide7 (117 mg, 0.303 mmol) and NMM (152 mg, 1.51 mmol). The reaction mixturewas stirred for 4 h at 60° C., 2 h at 65° C., and 1 h at 70° C. Afterconcentration, the residue was purified by column chromatography (silicagel, 10:1 CH₂Cl₂/MeOH, 5:1:0.1 CHCl₃/MeOH/NH₄OH) to afford compound 132(800 mg, 17% over 3 steps) as a yellow solid: ESI-MS m/z 765[C₇₂H₁₂₁ClN₁₈O₁₆+2H]²⁺.

Preparation of Hydrochloride Salt of(2R,2′R)—N,N′-(3,3′-(2-(4-(4-(3-(3,5-diamino-6-chloropyrazine-2-carbonyl)guanidino)butyl)-5,6,7,8-tetrahydronaphthalen-1-yloxy)ethylazanediyl)bis(propane-3,1-diyl))bis(2-amino-6-guanidinohexanamide)Compound 133

A solution of compound 132 (800 mg, 0.52 mmol) in EtOH (1.0 mL) wascharged with 4 N HCl (5.0 mL) and the reaction mixture was stirred for 4h at room temperature. The reaction mixture was concentrated and theresidue was redissolved in fresh 4 N HCl (5.0 mL). The reaction mixturewas stirred for 4 h at room temperature. After concentration, theresidue was purified by reverse-phase column chromatography andlyophilized to afford hydrochloric acid salt 133 (180 mg, 42%) as ayellow hygroscopic solid: ¹H NMR (400 MHz, D₂O) δ 7.04 (d, J=8.4 Hz,1H), 6.74 (d. J=8.4 Hz, 1H), 4.26 (br s, 2H), 3.88 (t, J=6.8 Hz, 2H),3.63 (br s, 2H), 3.32-3.27 (m, 10H), 3.06 (t, J=6.8 Hz, 4H), 2.65-2.50(m, 6H), 2.02-1.95 (m, 4H), 1.82-1.59 (m, 10H), 1.54-1.47 (m, 4H),1.36-1.30 (m, 4H).

Preparation of the Hydrochloride Salt of(2S,2′S)—N,N′-(3,3′-(3-(4-((S)-2-amino-3-(4-(4-(3-(3,5-diamino-6-chloropyrazine-2-carbonyl)guanidino)butyl)phenyl)propanamido)phenyl)propylazanediyl)bis(propane-3,1-diyl))bis(2-amino-6-guanidinohexanamide)-Compound145

Preparation of Compound 137

A solution of compound 135 (2.00 g, 9.26 mmol) in MeOH (10 mL) wascharged with NaCNBH₃ (2.00 g, 27.7 mmol) followed by AcOH (1.60 g, 27.7mmol) and compound 136 (4.79 g, 27.7 mmol). The reaction mixture wasstirred at room temperature for 24 h. Additional NaCNBH₃ (2.00 g, 27.7mmol), AcOH (1.60 g, 27.7 mmol), and compound 136 (3.20 g, 18.5 mmol)were added. After stirring at room temperature for 16 h, the solvent wasremoved. The residue was washed with 1 N Na₂CO₃ (30 mL) and purified bycolumn chromatography (silica gel, 10:1 CH₂Cl₂/MeOH) to afford compound137 (2.00 g, 44%) as a yellow oil: ¹H NMR (400 MHz, CDCl₃) δ 8.17 (d,J=8.6 Hz, 2H), 7.39 (d, J=8.6 Hz, 2H), 5.10 (br s, 2H), 3.22-3.18 (m,4H), 2.93 (br s, 5H), 2.81 (t, J=7.4 Hz, 2H), 2.03 (br s, 2H), 1.85 (brs, 5H), 1.42 (s, 18H).

Preparation of Compound 138

Compound 137 (2.00 g, 4.04 mmol) was dissolved in 4 N HCl in dioxane(100 mL) at room temperature and the reaction mixture was stirred atroom temperature for 2 h. After the solvent was removed, the residue waswashed with hexanes to afford compound 138 (1.20 g, 76%) as a whitesolid: ¹H NMR (400 MHz, CD₃OD) δ8.19 (d, J=8.6 Hz, 2H), 7.57 (d, J=8.6Hz, 2H), 3.69-3.62 (m, 1H), 3.36-3.32 (m, 4H), 3.08 (t, J=7.6 Hz, 4H),2.90 (t, J=7.6 Hz, 2H), 2.23-2.15 (m, 6H).

Preparation of Compound 140

A solution of compound 138 (100 mg, 0.248 mmol) in DMF (5.0 mL) wascharged with HATU (208 mg, 0.546 mmol) followed by compound 139 (242 mg,0.496 mmol) and DIPEA (128 mg, 0.992 mmol) at room temperature. Afterstirring at room temperature for 6 h, the solvent was removed and theresidue was purified by column chromatography (silica gel, 15:1CH₂Cl₂/MeOH) to afford compound 140 (90.0 mg, 29%) as a white solid: ¹HNMR (400 MHz, CD₃OD) δ 8.17 (d, J=8.6 Hz, 2H), 7.49 (d, J=8.6 Hz, 2H),3.95-3.92 (m, 2H), 3.75-3.69 (m, 1H), 3.35-3.32 (m, 4H), 3.26-3.19 (m,4H), 2.81 (br s, 4H), 1.98 (br s, 2H), 1.79-1.70 (m, 6H), 1.64-1.54 (m,8H), 1.51 (br s, 24H), 1.46-1.42 (m, 48H), 1.38-1.34 (m, 11H).

Preparation of Compound 141; SG-DVR-A-105

A suspension of compound 140 (100 mg, 0.081 mmol) and 10% Pd/C (50 mg)in EtOH (10 mL) was subjected to hydrogenation conditions (1 atm) for 8h at room temperature. The reaction mixture was filtered through celiteand washed with EtOH. The filtrate was concentrated and the residue waspurified by column chromatography (silica gel, 10:1 CH₂Cl₂/MeOH) toafford compound 141 (70 mg, 72%) as a white solid: ¹H NMR (300 MHz,CD₃OD) δ 6.97 (d, J=8.2 Hz, 2H), 6.67 (d, J=8.2 Hz, 2H), 3.94-3.90 (m,2H), 3.74-3.68 (m, 1H), 3.28-3.19 (m, 4H), 3.07 (br s, 6H), 2.58 (t,J=7.4 Hz, 2H), 1.98-1.94 (m, 2H), 1.87-1.78 (m, 4H), 1.74-1.56 (m, 8H),1.52 (s, 24H), 1.46 (s, 22H), 1.44 (s, 24H), 1.38-1.35 (m, 9H).

Preparation of Compound 142

A solution of compound 141 (150 mg, 0.124 mmol) in DMF (4.0 mL) wascharged with HATU (52 mg, 0.137 mmol) followed by compound 16 (58 mg,0.124 mmol) and DIPEA (63 mg, 0.496 mmol) at room temperature. Afterstirring at room temperature for 8 h, the solvent was removed and theresidue was purified by column chromatography (silica gel, 15:1CH₂Cl₂/MeOH) to afford compound 142 (130 mg, 63%) as a white solid: ¹HNMR (400 MHz, CD₃OD) δ 7.40 (d, J=8.6 Hz, 2H), 7.34-7.25 (m, 7H), 7.17(t, J=8.4 Hz, 4H), 5.08 (s, 2H), 4.39 (br s, 1H), 3.96-3.93 (m, 2H),3.35-3.31 (m, 8H), 3.24-3.19 (m, 5H), 3.12-3.07 (m, 1H), 2.94-2.89 (m,1H), 2.80-2.73 (m, 3H), 2.62-2.52 (m, 5H), 1.94-1.86 (m, 2H), 1.73 (brs, 6H), 1.61-1.54 (m, 7H), 1.51 (br s, 21H), 1.45 (br s, 44H), 1.38-1.34(m, 15H).

Preparation of Compound 143

A suspension of compound 142 (1.18 g, 0.713 mmol) and 10% Pd/C (120 mg)in EtOH (10 mL) was subjected to hydrogenation conditions (1 atm) for 8h at room temperature. The reaction mixture was filtered through celiteand washed with EtOH. The filtrate was concentrated to afford compound143 (680 mg, 62%) as a brown solid: ESI MS m/z 762 [C₇₇H₁₃₀N₁₄O₁₇+2H]²⁺.

Preparation of Compound 144

A solution of compound 143 (100 mg, 0.065 mmol) and methyl3,5-diamino-6-chloropyrazine-2-carbonylcarbamimidothioate hydroiodicacid salt (7, 30 mg, 0.078 mmol) in t-BuOH (10 mL) was charged withDIPEA (66 mg, 0.520 mmol) at room temperature. The reaction mixture washeated at 70° C. for 3 h and 80° C. for 2 h, cooled to room temperature,and concentrated in vacuum. The residue was purified by columnchromatography (silica gel, 10:1 CH₂Cl₂/MeOH, 4:1:0.1 CHCl₃/MeOH/NH₄OH)to afford compound 144 (40 mg, 35%) as a yellow solid: ¹H NMR (300 MHz,CD₃OD) δ 7.38 (d, J=8.2 Hz, 2H), 7.18-7.09 (m, 6H), 4.38 (br s, 1H),3.99 (br s, 2H), 3.63-3.53 (m, 1H), 3.36 (br s, 3H), 3.22 (br s, 4H),3.11-3.07 (m, 1H), 2.92-2.85 (m, 1H), 2.74 (t, J=7.2 Hz, 2H), 2.63-2.58(m, 4H), 2.46 (br s, 6H), 1.79-1.70 (m, 4H), 1.62-1.55 (m, 13H), 1.51(s, 19H), 1.46 (s, 9H), 1.43 (s, 20H), 1.38 (s, 9H).

Preparation of the Hydrochloride Salt of(2S,2′S)—N,N′-(3,3′-(3-(4-((S)-2-amino-3-(4-(4-(3-(3,5-diamino-6-chloropyrazine-2-carbonyl)guanidino)butyl)phenyl)propanamido)phenyl)propylazanediyl)bis(propane-3,1-diyl))bis(2-amino-6-guanidinohexanamide)-Compound145

A solution of compound 144 (250 mg, 0.144 mmol) in EtOH (3.0 mL) wascharged with 4 N aqueous HCl (25 mL) and the reaction mixture wasstirred for 6 h at room temperature. The reaction mixture wasconcentrated in vacuum, and the residue was purified by reverse-phasecolumn chromatography and lyophilized to afford hydrochloric acid salt145 (70 mg, 38%) as a yellow hygroscopic solid: ¹H NMR (400 MHz, D₂O) δ7.23 (d, J=8.4 Hz, 2H), 7.17 (d, J=8.4 Hz, 4H), 7.11 (d, J=8.4 Hz, 2H),4.26-4.23 (m, 1H), 3.92 (t, J=6.8 Hz, 2H), 3.35-3.28 (m, 2H), 3.27-3.18(m, 5H), 3.16-3.09 (m, 11H), 2.65-2.57 (m, 4H), 1.97-1.78 (m, 10H),1.70-1.63 (m, 2H), 1.60-1.51 (m, 6H), 1.39-1.33 (m, 4H). HRMS calculatedfor C₄₈H₈₀ClN₂₀O₄[M+H]⁺, 1035.6354. found 1035.6375.

All of the references cited above are incorporated herein by reference.In the event of a conflict between the foregoing description and areference, the description provided herein controls.

What is claimed is:
 1. A compound represented by formula (I):

and racemates, enantiomers, diastereomers, tautomers, polymorphs,pseudopolymorphs and pharmaceutically acceptable salts, thereof,wherein: X is hydrogen, halogen, trifluoromethyl, lower alkyl,unsubstituted or substituted phenyl, lower alkyl-thio, phenyl-loweralkyl-thio, lower alkyl-sulfonyl, or phenyl-lower alkyl-sulfonyl; Y ishydrogen, hydroxyl, mercapto, lower alkoxy, lower alkyl-thio, halogen,lower alkyl, unsubstituted or substituted mononuclear aryl, or —N(R²)₂;R¹ is hydrogen or lower alkyl; each R² is, independently, —R⁷,—(CH₂)_(m)—OR⁸, —(CH₂)_(m)—NR⁷R¹⁰, —(CH₂)_(n)(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸,—(CH₂CH₂O)_(m)—R⁸, —(CH₂CH₂O)_(m)—CH₂CH₂NR⁷R¹⁰, —(CH₂)_(n)—C(═O)NR⁷R¹⁰,—(CH₂)_(n)-(Z)_(g)—R⁷, —(CH₂)_(m)—NR¹⁰—CH₂(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸,—(CH₂)_(n)—CO₂R⁷, or

R³ and R⁴ are each, independently, hydrogen, lower alkyl, hydroxyl-loweralkyl, phenyl, (phenyl)-lower alkyl, (halophenyl)-lower alkyl,((lower-alkyl)phenyl)-lower-alkyl, ((lower-alkoxy)phenyl)-lower-alkyl,(naphthyl)-lower-alkyl, or (pyridyl)-lower-alkyl, or a group representedby formula A or formula B, with the proviso that at least one of R³ andR⁴ is a group represented by the formula A or formula B;—(C(R^(L))₂)_(o)-x-(C(R^(L))₂)_(p)A¹  formula A:—(C(R^(L))₂)_(o)-x-(C(R^(L))₂)_(p)A²  formula B: A¹ is a C₆-C₁₅-memberedaromatic carbocycle substituted with at least one R⁵ and the remainingsubstituents are R⁶; A² is a six to fifteen-membered aromaticheterocycle substituted with at least one R⁵ and the remainingsubstituents are R⁶ wherein said aromatic heterocycle comprises 1-4heteroatoms selected from the group consisting of O, N, and S; eachR^(L) is, independently, —R⁷, —(CH₂)_(n)—OR⁸, —O—(CH₂)_(m)—OR⁸,—(CH₂)_(n)—NR⁷R¹⁰, —O—(CH₂)_(m)—NR⁷R¹⁰,—(CH₂)_(n)(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸,—O—(CH₂)_(m)(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸, —(CH₂CH₂O)_(m)—R⁸,—O—(CH₂CH₂O)_(m)—R⁸, —(CH₂CH₂O)_(m)—CH₂CH₂NR⁷R¹⁰,—O—(CH₂CH₂O)_(m)—CH₂CH₂NR⁷R¹⁰, —(CH₂)_(n)—C(═O)NR⁷R¹⁰,—O—(CH₂)_(m)—C(═O)NR⁷R¹⁰, —(CH₂)_(n)-(Z)_(g)—R⁷,—O—(CH₂)_(m)-(Z)_(g)—R⁷, —(CH₂)_(n)—NR¹⁰—CH₂(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸,—O—(CH₂)_(m)—NR¹⁰—CH₂(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸, —(CH₂)_(n)—CO₂R⁷,—O—(CH₂)_(m)—CO₂R⁷, —OSO₃H, —O-glucuronide, —O-glucose,

each o is, independently, an integer from 0 to 10; each p is,independently, an integer from 0 to 10; with the proviso that the sum ofo and p in each contiguous chain is from 1 to 10; each x is,independently, O, NR¹⁰, C(═O), CHOH, C(═N—R¹⁰), CHNR⁷R¹⁰, or a singlebond; each R⁵ is, independently, -Link-(CH₂)_(m)-CAP,-Link-(CH₂)_(n)(CHOR⁸)(CHOR⁸)_(n)-CAP, -Link-(CH₂CH₂O)_(m)—CH₂-CAP,-Link-(CH₂CH₂O)_(m)—CH₂CH₂-CAP, -Link-(CH₂)_(m)-(Z)_(g)-CAP,-Link-(CH₂)_(n)(Z)_(g)—(CH₂)_(m)-CAP,-Link-(CH₂)_(n)—NR¹³—CH₂(CHOR⁸)(CHOR⁸)_(n)-CAP,-Link-(CH₂)_(n)—(CHOR⁸)_(m)CH₂—NR¹³-(Z)_(g)-CAP,-Link-(CH₂)_(n)NR¹³—(CH₂)_(m)(CHOR⁸)_(n)CH₂NR¹³—(Z)_(g)-CAP,-Link-(CH₂)_(m)-(Z)_(g)—(CH₂)_(m)-CAP, -Link-NH—C(═O)—NH—(CH₂)_(m)-CAP,-Link-(CH₂)_(m)—C(═O)NR¹³—(CH₂)_(m)-CAP,-Link-(CH₂)_(n)-(Z)_(g)—(CH₂)_(m)-(Z)_(g)-CAP, or-Link-Z_(g)-(CH₂)_(m)-Het-(CH₂)_(m)-CAP; each R⁶ is, independently, R⁵,—R⁷, —OR¹¹, —N(R⁷)₂, —(CH₂)_(m)—OR⁸, —O—(CH₂)_(m)—OR⁸,—(CH₂)_(n)—NR⁷R¹⁰, —O—(CH₂)_(m)—NR⁷R¹⁰,—(CH₂)_(n)(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸,—O—(CH₂)_(m)(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸, —(CH₂CH₂O)_(m)—R⁸,—O—(CH₂CH₂O)_(m)—R⁸, —(CH₂CH₂O)_(m)—CH₂CH₂NR⁷R¹⁰,—O—(CH₂CH₂O)_(m)—CH₂CH₂NR⁷R¹⁰, —(CH₂)_(n)—C(═O)NR⁷R¹⁰,—O—(CH₂)_(m)—C(═O)NR⁷R¹⁰, —(CH₂)_(n)-(Z)_(g)—R⁷,—O—(CH₂)_(m)-(Z)_(g)—R⁷, —(CH₂)_(n)—NR¹⁰—CH₂(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸,—O—(CH₂)_(m)—NR¹⁰—CH₂(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸, —(CH₂)_(n)—CO₂R⁷,—O—(CH₂)_(m)—CO₂R⁷, —OSO₃H, —O-glucuronide, —O-glucose,

wherein when two R⁶ are —OR¹¹ and are located adjacent to each other onthe aromatic carbocycle or aromatic heterocycle, the two OR¹¹ may form amethylenedioxy group: each R⁷ is, independently, hydrogen, lower alkyl,phenyl, substituted phenyl or —CH₂(CHOR⁸)_(m)—CH₂OR⁸; each R⁸ is,independently, hydrogen, lower alkyl, —C(═O)—R¹¹, glucuronide,2-tetrahydropyranyl, or

each R⁹ is, independently, —CO₂R⁷, —CON(R⁷)₂, —SO₂CH₃, —C(═O)R⁷,—CO₂R¹³, —CON(R¹³)₂, —SO₂CH₂R¹³, or —C(═O)R¹³; each R¹⁰ is,independently, —H, —SO₂CH₃, —CO₂R⁷, —C(═O)NR⁷R⁹, —C(═O)R⁷, or—CH₂—(CHOH)_(n)—CH₂OH; each Z is, independently, —(CHOH)—, —C(═O)—,—(CHNR⁷R¹⁰)—, —(C═NR¹⁰)—, —NR¹⁰—, —(CH₂)_(n)—, —(CHNR¹³R¹³)—,—(C═NR¹³)—, or —NR¹³—; each R¹¹ is, independently, hydrogen, loweralkyl, phenyl lower alkyl or substituted phenyl lower alkyl; each R¹²is, independently, —SO₂CH₃, —CO₂R⁷, —C(═O)NR⁷R⁹, —C(═O)R⁷,—CH₂(CHOH)_(n)—CH₂OH, —CO₂R, —C(═O)NR⁷R⁷, or —C(═O)R⁷; each R¹³ is,independently, hydrogen, —OR⁷, R¹⁰, R¹¹ or R¹²; each g is,independently, an integer from 1 to 6; each m is, independently, aninteger from 1 to 7; each n is, independently, an integer from 0 to 7;each -Het- is, independently, —N(R⁷)—, —N(R¹⁰)—, —S—, —SO—, —SO₂—; —O—,—SO₂NH—, —NHSO₂—, —NR⁷CO—, —CONR⁷—, —N(R¹³)—, —SO₂NR¹³—, —NR¹³CO—, or—CONR¹³—; each Link is, independently, —O—, —(CH₂)_(n)—, —O(CH₂)_(m)—,—NR¹³—C(═O)—NR¹³—, —NR¹³—C(═O)—(CH₂)_(m)—, —C(═O)NR¹³—(CH₂)_(m) ⁻,—(CH₂)_(n)-(Z)_(g)—(CH₂)_(n)—, —S—, —SO—, —SO₂—, —SO₂NR⁷—, —SO₂NR¹⁰—, or-Het-; each CAP is